Lymphangiogenesis guidance by paracrine and pericellular factors.

Lymphatic vessels are important for tissue fluid homeostasis, lipid absorption, and immune cell trafficking and are involved in the pathogenesis of several human diseases. The mechanisms by which the lymphatic vasculature network is formed, remodeled, and adapted to physiological and pathological challenges are controlled by an intricate balance of growth factor and biomechanical cues. These transduce signals for the readjustment of gene expression and lymphatic endothelial migration, proliferation, and differentiation. In this review, we describe several of these cues and how they are integrated for the generation of functional lymphatic vessel networks.

A closer look at adipose tissue lymphatics and their markers.

PURPOSE OF REVIEW: Lymphatic vessels are found in most tissues, with the exception of the cornea and the central nervous system. Tissues that have high exposure to antigens, such as the skin and the intestine, have especially extensive lymphatic vascular networks. Despite being densely vascularized with blood vessels, adipose tissue is poorly permeated with lymphatic vasculature. Here, we focus on the recent advances in the research on adipose tissue lymphatics and present a lymphatic-focused analysis of published single-cell and single-nucleus RNA sequencing datasets of adipose tissues. RECENT FINDINGS: Although lymphatic expansion in obesity may limit inflammation and promote glycerol efflux from adipose tissue, lymphatic endothelial cells (LECs) secrete factors that reduce brown adipocyte thermogenesis. Transcriptomic analyses of these cells show that they express common lymphatic markers such as Proxl, but datasets from different studies show great variation in gene expression values due to the low number of captured LECs, depot differences, and species-specific gene expression patterns. SUMMARY: As the importance of LECs in the homeostasis of adipose tissue has become evident, investigators want to shed light on the specific interactions of lymphatics with other cell types in adipose tissues. Extracting LECs from readily available transcriptomics datasets provides a standpoint for investigators for future research. However, systematic studies are needed to reveal unique identities according to depot and species-specific LEC signatures.

VEGF-C/VEGFR-3 signalling in macrophages ameliorates acute lung injury.

BACKGROUND: Successful recovery from acute lung injury requires inhibition of neutrophil influx and clearance of apoptotic neutrophils. However, the mechanisms underlying recovery remain unclear. We investigated the ameliorative effects of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR-3) signalling in macrophages in lipopolysaccharide (LPS)-induced lung injury. METHODS: LPS was intranasally injected into wild-type and transgenic mice. Gain and loss of VEGF-C/VEGFR-3 signalling function experiments employed adenovirus-mediated intranasal delivery of VEGF-C (Ad-VEGF-C vector) and soluble VEGFR-3 (sVEGFR-3) or anti-VEGFR-3 blocking antibodies and mice with a deletion of VEGFR-3 in myeloid cells. RESULTS: The early phase of lung injury was significantly alleviated by the overexpression of VEGF-C with increased levels of bronchoalveolar lavage (BAL) fluid interleukin-10 (IL-10), but worsened in the later phase by VEGFR-3 inhibition upon administration of Ad-sVEGFR-3 vector. Injection of anti-VEGFR-3 antibodies to mice in the resolution phase inhibited recovery from lung injury. The VEGFR-3-deleted mice had a shorter survival time than littermates and more severe lung injury in the resolution phase. Alveolar macrophages in the resolution phase digested most of the extrinsic apoptotic neutrophils and VEGF-C/VEGFR-3 signalling increased efferocytosis via upregulation of integrin αv in the macrophages. We also found that incubation with BAL fluid from acute respiratory distress syndrome (ARDS) patients, but not from controls, decreased VEGFR-3 expression and the efficiency of IL-10 expression and efferocytosis in human monocyte-derived macrophages. CONCLUSIONS: VEGF-C/VEGFR-3 signalling in macrophages ameliorates experimental lung injury. This mechanism may also provide an explanation for ARDS resolution.

Vascular endothelial growth factor signaling in development and disease.

Vascular endothelial growth factors (VEGFs) are best known for their involvement in orchestrating the development and maintenance of the blood and lymphatic vascular systems. VEGFs are secreted by a variety of cells and they bind to their cognate tyrosine kinase VEGF receptors (VEGFRs) in endothelial cells to elicit various downstream effects. In recent years, there has been tremendous progress in elucidating different VEGF/VEGFR signaling functions in both the blood and lymphatic vascular systems. Here, and in the accompanying poster, we present key elements of the VEGF/VEGFR pathway and highlight the classical and newly discovered functions of VEGF signaling in blood and lymphatic vessel development and pathology.

Endothelial cell-derived semaphorin 3A inhibits filopodia formation by blood vascular tip cells.

Vascular endothelial growth factor (VEGF)-A is a well-known major chemoattractant driver of angiogenesis--the formation of new blood vessels from pre-existing ones. However, the repellent factors that fine-tune this angiogenic process remain poorly characterized. We investigated the expression and functional role of endothelial cell-derived semaphorin 3A (Sema3A) in retinal angiogenesis, using genetic mouse models. We found Sema3a mRNA expression in the ganglion cell layer and the presence of Sema3A protein on larger blood vessels and at the growing front of blood vessels in neonatal retinas. The Sema3A receptors neuropilin-1 and plexin-A1 were expressed by retinal blood vessels. To study the endothelial cell-specific role of Sema3A, we generated endothelial cell-specific Sema3A knockout mouse strains by constitutive or inducible vascular endothelial cadherin-Cre-mediated gene disruption. We found that in neonatal retinas of these mice, both the number and the length of tip cell filopodia were significantly increased and the leading edge growth pattern was irregular. Retinal explant experiments showed that recombinant Sema3A significantly decreased VEGF-A-induced filopodia formation. Endothelial cell-specific knockout of Sema3A had no impact on blood vessel density or skin vascular leakage in adult mice. These findings indicate that endothelial cell-derived Sema3A exerts repelling functions on VEGF-A-induced tip cell filopodia and that a lack of this signaling cannot be rescued by paracrine sources of Sema3A.

VEGFR3 Modulates Vascular Permeability by Controlling VEGF/VEGFR2 Signaling.

RATIONALE: Vascular endothelial growth factor (VEGF) is the main driver of angiogenesis and vascular permeability via VEGF receptor 2 (VEGFR2), whereas lymphangiogenesis signals are transduced by VEGFC/D via VEGFR3. VEGFR3 also regulates sprouting angiogenesis and blood vessel growth, but to what extent VEGFR3 signaling controls blood vessel permeability remains unknown. OBJECTIVE: To investigate the role of VEGFR3 in the regulation of VEGF-induced vascular permeability. METHODS AND RESULTS: Long-term global Vegfr3 gene deletion in adult mice resulted in increased fibrinogen deposition in lungs and kidneys, indicating enhanced vascular leakage at the steady state. Short-term deletion of Vegfr3 in blood vascular endothelial cells increased baseline leakage in various tissues, as well as in tumors, and exacerbated vascular permeability in response to VEGF, administered via intradermal adenoviral delivery or through systemic injection of recombinant protein. VEGFR3 gene silencing upregulated VEGFR2 protein levels and phosphorylation in cultured endothelial cells. Consistent with elevated VEGFR2 activity, vascular endothelial cadherin showed reduced localization at endothelial cell-cell junctions in postnatal retinas after Vegfr3 deletion, or after VEGFR3 silencing in cultured endothelial cells. Furthermore, concurrent deletion of Vegfr2 prevented VEGF-induced excessive vascular leakage in mice lacking Vegfr3. CONCLUSIONS: VEGFR3 limits VEGFR2 expression and VEGF/VEGFR2 pathway activity in quiescent and angiogenic blood vascular endothelial cells, thereby preventing excessive vascular permeability.

Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

Lymphatic System in Cardiovascular Medicine.

The mammalian circulatory system comprises both the cardiovascular system and the lymphatic system. In contrast to the blood vascular circulation, the lymphatic system forms a unidirectional transit pathway from the extracellular space to the venous system. It actively regulates tissue fluid homeostasis, absorption of gastrointestinal lipids, and trafficking of antigen-presenting cells and lymphocytes to lymphoid organs and on to the systemic circulation. The cardinal manifestation of lymphatic malfunction is lymphedema. Recent research has implicated the lymphatic system in the pathogenesis of cardiovascular diseases including obesity and metabolic disease, dyslipidemia, inflammation, atherosclerosis, hypertension, and myocardial infarction. Here, we review the most recent advances in the field of lymphatic vascular biology, with a focus on cardiovascular disease.

High-Fat Diet in the Absence of Obesity Does Not Aggravate Surgically Induced Lymphoedema in Mice.

BACKGROUND: Lymphoedema represents the cardinal manifestation of lymphatic dysfunction and is associated with expansion of the adipose tissue in the affected limb. In mice, high-fat diet (HFD)-induced obesity was associated with impaired collecting lymphatic vessel function, and adiposity aggravated surgery-induced lymphoedema in a mouse model. The aim of the current study was to investigate whether adiposity is necessary to impair lymphatic function or whether increased lipid exposure alone might be sufficient in a surgical lymphoedema model. METHODS: To investigate the role of increased lipid exposure in lymphoedema development we used a well-established mouse tail lymphoedema model. Female mice were subjected to a short-term (6 weeks) HFD, without development of obesity, before surgical induction of lymphedema. Lymphoedema was followed over a period of 6 weeks measuring oedema, evaluating tissue histology and lymphatic vascular function. RESULTS: HFD increased baseline angiogenesis and average lymphatic vessel size in comparison to the chow control group. Upon induction of lymphedema, HFD-treated mice did not exhibit aggravated oedema and no morphological differences were observed in the blood and lymphatic vasculature. Importantly, the levels of fibro-adipose tissue deposition were comparable between the 2 groups and lymphatic vessel function was not impaired as a result of the HFD. Although the net immune cell infiltration was comparable, the HFD group displayed an increased infiltration of macrophages, which exhibited an M2 polarization phenotype. CONCLUSIONS: These results indicate that increased adiposity rather than dietary influences determines predisposition to or severity of lymphedema.

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.

Decline of lymphatic vessel density and function in murine skin during aging.

Lymphatic vessels play important roles in the pathogenesis of many conditions that have an increased prevalence in the elderly population. However, the effects of the aging process on the lymphatic system are still relatively unknown. We have applied non-invasive imaging and whole-mount staining techniques to assess the lymphatic vessel function and morphology in three different age groups of mice: 2 months (young), 7 months (middle-aged), and 18 months (aged). We first developed and validated a new method to quantify lymphatic clearance from mouse ear skin, using a lymphatic-specific near-infrared tracer. Using this method, we found that there is a prominent decrease in lymphatic vessel function during aging since the lymphatic clearance was significantly delayed in aged mice. This loss of function correlated with a decreased lymphatic vessel density and a reduced lymphatic network complexity in the skin of aged mice as compared to younger controls. The blood vascular leakage in the skin was slightly increased in the aged mice, indicating that the decreased lymphatic function was not caused by a reduced capillary filtration in aged skin. The decreased function of lymphatic vessels with aging might have implications for the pathogenesis of a number of aging-related diseases.

The role of neuropilin-1/semaphorin 3A signaling in lymphatic vessel development and maturation.

During development, the lymphatic and the blood vascular system form highly branched networks that show extensive architectural similarities with the peripheral nervous system. Increasing evidence suggests that the vascular and the nervous systems share signaling pathways to overcome common challenges such as guidance of growth and patterning. Semaphorins, a large group of proteins originally identified as axon guidance molecules with repelling function, and their receptors, neuropilins and plexins, have recently also been implicated in vascular development. Here, we summarize the role of semaphorins and their receptors in angiogenesis and lymphangiogenesis, with an emphasis on neuropilin-1/semaphorin 3A interactions in lymphatic vessel maturation and valve formation. Understanding the basic principles of lymphatic vessel development and maturation might facilitate the development of therapies for the treatment of human diseases associated with lymphedema.

Novel role for ALCAM in lymphatic network formation and function.

Adhesion molecules play an important role in vascular biology because they mediate vascular stability, permeability, and leukocyte trafficking to and from tissues. Performing microarray analyses, we have recently identified activated leukocyte cell adhesion molecule (ALCAM) as an inflammation-induced gene in lymphatic endothelial cells (LECs). ALCAM belongs to the immunoglobulin superfamily and engages in homophilic as well as heterophilic interactions. In this study, we found ALCAM to be expressed at the protein level in human and murine lymphatic and blood vascular endothelial cells in vitro and in the vasculature of human and murine tissues in vivo. Functional in vitro experiments revealed that ALCAM mediates adhesive interactions, migration, and tube formation in LECs, suggesting a role for ALCAM in lymphatic vessel (LV) stability and in lymphangiogenesis. Furthermore, ALCAM supported dendritic cell (DC) adhesion to lymphatic endothelium. In agreement with these findings, experiments performed in ALCAM mice detected reduced LEC numbers in various tissues and defects in the formation of an organized LV network. Moreover, DC migration from lung to draining lymph nodes was compromised in ALCAM mice. Collectively, our data reveal a novel role for ALCAM in stabilizing LEC-LEC interactions and in the organization and function of the LV network.

An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation.

RATIONALE: Lymphatic vasculature plays important roles in tissue fluid homeostasis maintenance and in the pathology of human diseases. Yet, the molecular mechanisms that control lymphatic vessel maturation remain largely unknown. OBJECTIVE: We analyzed the gene expression profiles of ex vivo isolated lymphatic endothelial cells to identify novel lymphatic vessel expressed genes and we investigated the role of semaphorin 3A (Sema3A) and neuropilin-1 (Nrp-1) in lymphatic vessel maturation and function. METHODS AND RESULTS: Lymphatic and blood vascular endothelial cells from mouse intestine were isolated using fluorescence-activated cell sorting, and transcriptional profiling was performed. We found that the axonal guidance molecules Sema3A and Sema3D were highly expressed by lymphatic vessels. Importantly, we found that the semaphorin receptor Nrp-1 is expressed on the perivascular cells of the collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1 but not with an antibody that blocks VEGF-A binding to Nrp-1 resulted in a complex phenotype of impaired lymphatic vessel function, enhanced perivascular cell coverage, and abnormal lymphatic vessel and valve morphology. CONCLUSIONS: Together, these results reveal an unanticipated role of Sema3A-Nrp-1 signaling in the maturation of the lymphatic vascular network likely via regulating the perivascular cell coverage of the vessels thus affecting lymphatic vessel function and lymphatic valve development.

DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Whole-mount immunofluorescence staining of blood and lymphatic vessels in murine small intestine.

The small intestine is an excellent model for studying changes in vasculature in response to different diseases or gene deletions. Here, we present a protocol for whole-mount immunofluorescence staining of blood and lymphatic vessels in the adult mouse small intestine. We describe the steps for perfusion fixation, tissue sample preparation, immunofluorescence staining, and whole-mount preparation of stained samples. Our protocol will enable researchers to visualize and analyze the intricate network of vessels in the small intestine. For complete details on the use and execution of this protocol, please refer to Karaman et al. (2022).1.

Blockade of VEGFR3 signaling leads to functional impairment of dural lymphatic vessels without affecting autoimmune neuroinflammation.

The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II-associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium.

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Bexmarilimab-induced macrophage activation leads to treatment benefit in solid tumors: The phase I/II first-in-human MATINS trial.

Macrophage Clever-1 contributes to impaired antigen presentation and suppression of anti-tumor immunity. This first-in-human trial investigates the safety and tolerability of Clever-1 blockade with bexmarilimab in patients with treatment-refractory solid tumors and assesses preliminary anti-tumor efficacy, pharmacodynamics, and immunologic correlates. Bexmarilimab shows no dose-limiting toxicities in part I (n = 30) and no additional safety signals in part II (n = 108). Disease control (DC) rates of 25%-40% are observed in cutaneous melanoma, gastric, hepatocellular, estrogen receptor-positive breast, and biliary tract cancers. DC associates with improved survival in a landmark analysis and correlates with high pre-treatment intratumoral Clever-1 positivity and increasing on-treatment serum interferon γ (IFNγ) levels. Spatial transcriptomics profiling of DC and non-DC tumors demonstrates bexmarilimab-induced macrophage activation and stimulation of IFNγ and T cell receptor signaling selectively in DC patients. These data suggest that bexmarilimab therapy is well tolerated and show that macrophage targeting can promote immune activation and tumor control in late-stage cancer.