The complex TIE between macrophages and angiogenesis

Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the process of new blood vessel formation from the pre-existing vasculature. Macrophages play crucial roles at each step of the angiogenic cascade, starting from new blood vessel sprouting to the remodelling of the vascular plexus and vessel maturation. Macrophages form promising targets for both pro- and anti-angiogenic treatments. However, to target macrophages, we will first need to understand the mechanisms that control the functional plasticity of macrophages during each of the steps of the angiogenic cascade. Here, we review recent insights in this topic. Special attention will be given to the TIE2-expressing macrophage (TEM), which is a subtype of highly angiogenic macrophages that is able to influence angiogenesis via the angiopoietin-TIE pathway

The Neurotrophic Receptor Ntrk2 Directs Lymphoid Tissue Neovascularization during Leishmania donovani Infection

The neurotrophic tyrosine kinase receptor type 2 (Ntrk2, also known as TrkB) and its ligands brain derived neurotrophic factor (Bdnf), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) are known primarily for their multiple effects on neuronal differentiation and survival. Here, we provide evidence that Ntrk2 plays a role in the pathologic remodeling of the spleen that accompanies chronic infection. We show that in Leishmania donovani-infected mice, Ntrk2 is aberrantly expressed on splenic endothelial cells and that new maturing blood vessels within the white pulp are intimately associated with F4/80hiCD11bloCD11c+ macrophages that express Bdnf and NT-4/5 and have pro-angiogenic potential in vitro. Furthermore, administration of the small molecule Ntrk2 antagonist ANA-12 to infected mice significantly inhibited white pulp neovascularization but had no effect on red pulp vascular remodeling. We believe this to be the first evidence of the Ntrk2/neurotrophin pathway driving pathogen-induced vascular remodeling in lymphoid tissue. These studies highlight the therapeutic potential of modulating this pathway to inhibit pathological angiogenesis

Intravital Two-Photon Microscopy of Immune Cell Dynamics in Corneal Lymphatic Vessels

BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions

Novel Characterization of Lymphatic Valve Formation during Corneal Inflammation

Lymphatic research has progressed rapidly in recent years. Though lymphatic dysfunction has been found in a wide array of disorders from transplant rejection to cancer metastasis, to date, there is still little effective treatment for lymphatic diseases. The cornea offers an optimal site for lymphatic research due to its accessible location, transparent nature, and lymphatic-free but inducible features. However, it still remains unknown whether lymphatic valves exist in newly formed lymphatic vessels in the cornea, and how this relates to an inflammatory response. In this study, we provide the first evidence showing that lymphatic valves were formed in mouse cornea during suture-induced inflammation with the up-regulation of integrin alpha 9. The number of corneal valves increased with the progression of inflammatory lymphangiogenesis. Moreover, we have detected lymphatic valves at various developmental stages, from incomplete to more developed ones. In addition to defining the average diameter of lymphatic vessels equipped with lymphatic valves, we also report that lymphatic valves were more often located near the branching points. Taken together, these novel findings not only provide new insights into corneal lymphatic formation and maturation, but also identify a new model for future investigation on lymphatic valve formation and possibly therapeutic intervention

Transient postoperative vascular endothelial growth factor (VEGF)-neutralisation improves graft survival in corneas with partly regressed inflammatory neovascularisation

Background: High-risk keratoplasties are usually performed after an uninflamed and quiescent interval in corneas with partly regressed blood and lymphatic vessels. We analysed whether the inhibition of post-keratoplasty revascularisation in mice with partly regressed corneal vessels (“intermediate-risk”) improves graft survival. Methods: Three interrupted stromal sutures (11-0) in corneas of Balb/c mice (6–8 weeks old) were placed for 6 weeks. Six months after suture removal, penetrating keratoplasty was performed with C57BL/6 donors. The treatment group received a vascular endothelial growth factor-A specific cytokine trap (VEGF Trap) intraperitoneally at days 0, 4, 7 and 14 after keratoplasty (25 mg/kg per mouse; controls received equal amounts of Fc protein). Pathological haemangiogenesis and lymphangiogenesis prior to as well as 3 days or 8 weeks after keratoplasty and graft survival were analysed. Results: Three days after keratoplasty corneal revascularisation was sufficiently reduced by VEGF Trap (haem-vascularised areas 42.7% reduction; lymph-vascularised areas 54.7% reduction). Survival proportions 8 weeks after keratoplasty were 36% in the treatment group compared with 9% in the control group (n = 11; p<0.05). At that time no differences in haemangiogenesis or lymphangiogenesis were observed between the two groups. Conclusion: Early transient postoperative induction of haemangiogenesis and lymphangiogenesis and reformation of regressed corneal blood and lymphatic vessels are important for transplant rejections after “intermediate-risk” corneal transplantation

The Herpes Simplex Virus-1 Transactivator Infected Cell Protein-4 Drives VEGF-A Dependent Neovascularization

Herpes simplex virus-1 (HSV-1) causes lifelong infection affecting between 50 and 90% of the global population. In addition to causing dermal lesions, HSV-1 is a leading cause of blindness resulting from recurrent corneal infection. Corneal disease is characterized by loss of corneal immunologic privilege and extensive neovascularization driven by vascular endothelial growth factor-A (VEGF-A). In the current study, we identify HSV-1 infected cells as the dominant source of VEGF-A during acute infection, and VEGF-A transcription did not require TLR signaling or MAP kinase activation. Rather than being an innate response to the pathogen, VEGF-A transcription was directly activated by the HSV-1 encoded immediate early transcription factor, ICP4. ICP4 bound the proximal human VEGF-A promoter and was sufficient to promote transcription. Transcriptional activation also required cis GC-box elements common to the VEGF-A promoter and HSV-1 early genes. Our results suggest that the neovascularization characteristic of ocular HSV-1 disease is a direct result of HSV-1's major transcriptional regulator, ICP4, and similarities between the VEGF-A promoter and those of HSV-1 early genes

Myeloid Cells Contribute to Tumor Lymphangiogenesis

The formation of new blood vessels (angiogenesis) and lymphatic vessels (lymphangiogenesis) promotes tumor outgrowth and metastasis. Previously, it has been demonstrated that bone marrow-derived cells (BMDC) can contribute to tumor angiogenesis. However, the role of BMDC in lymphangiogenesis has largely remained elusive. Here, we demonstrate by bone marrow transplantation/reconstitution and genetic lineage-tracing experiments that BMDC integrate into tumor-associated lymphatic vessels in the Rip1Tag2 mouse model of insulinoma and in the TRAMP-C1 prostate cancer transplantation model, and that the integrated BMDC originate from the myelomonocytic lineage. Conversely, pharmacological depletion of tumor-associated macrophages reduces lymphangiogenesis. No cell fusion events are detected by genetic tracing experiments. Rather, the phenotypical conversion of myeloid cells into lymphatic endothelial cells and their integration into lymphatic structures is recapitulated in two in vitro tube formation assays and is dependent on fibroblast growth factor-mediated signaling. Together, the results reveal that myeloid cells can contribute to tumor-associated lymphatic vessels, thus extending the findings on the previously reported role of hematopoietic cells in lymphatic vessel formation

Lymphatic density and metastatic spread in human malignant melanoma

Lymphatic density and metastatic spread in human malignant melanoma. Malignant melanoma (MM), the most common cause of skin cancer deaths, metastasises to regional lymph nodes. In animal models of other cancers, lymphatic growth is associated with metastasis. To assess if lymphatic density (LD) was increased in human MM, and its association with metastasis, we measured LD inside and around archival MM samples (MM, n = 21), and compared them with normal dermis (n = 11), basal cell carcinoma (BCC, n = 6) and Merkel cell carcinoma (MCC), a skin tumour thought to metastasise through a vascular route (MCC, n = 6). Lymphatic capillary density (mm(-2)), as determined by immunohistochemical staining with the lymphatic specific marker LYVE-1, was significantly increased around MM (10.0+/-2.5 mm(-2)) compared with normal dermis (2.4+/-0.9 mm(-2)), BCC (3.0+/-0.9 mm(-2)) and MCC (2.4+/-1.4 mm(-2)) (P<0.0001). There was a small decrease in LD inside MM (1.1+/-0.7 mm(-2)) compared with normal dermis, but a highly significant decrease in BCC (0.14+/-0.13) and MCC (0.12+/-2.4) (P<0.01 Kruskal-Wallis). Astonishingly, LD discriminated between melanomas that subsequently metastasised (12.8+/-1.6 mm(-2)) and those that did not (5.4+/-1.1 mm(-2), P<0.01, Mann-Whitney). Lymphatic invasion by tumour cells was seen mainly in MM that metastasised (70% compared with 12% not metastasising, P<0.05 Fisher's Exact test). The results show that LD was increased around MMs, and that LD and tumour cell invasion of lymphatics may help to predict metastasis. To this end, a prognostic index was calculated using LD, lymphatic invasion and thickness that clearly discriminated metastatic from nonmetastatic tumours

The role of the VEGF-C/VEGFR-3 axis in cancer progression

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3) (also called VEGFR-3) is activated by its specific ligand, VEGF-C, which promotes cancer progression. The VEGF-C/VEGFR-3 axis is expressed not only by lymphatic endothelial cells but also by a variety of human tumour cells. Activation of the VEGF-C/VEGFR-3 axis in lymphatic endothelial cells can facilitate metastasis by increasing the formation of lymphatic vessels (lymphangiogenesis) within and around tumours. The VEGF-C/VEGFR-3 axis plays a critical role in leukaemic cell proliferation, survival, and resistance to chemotherapy. Moreover, activation of the VEGF-C/VEGFR-3 axis in several types of solid tumours enhances cancer cell mobility and invasion capabilities, promoting cancer cell metastasis. In this review, we discuss the novel function and molecular mechanism of the VEGF-C/VEGFR-3 axis in cancer progression