Georgia Zarkada

Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a key process in several pathological conditions, including tumour growth and age-related macular degeneration. Vascular endothelial growth factors (VEGFs) stimulate angiogenesis and lymphangiogenesis by activating VEGF receptor (VEGFR) tyrosine kinases in endothelial cells. VEGFR-3 (also known as FLT-4) is present in all endothelia during development, and in the adult it becomes restricted to the lymphatic endothelium. However, VEGFR-3 is upregulated in the microvasculature of tumours and wounds. Here we demonstrate that VEGFR-3 is highly expressed in angiogenic sprouts, and genetic targeting of VEGFR-3 or blocking of VEGFR-3 signalling with monoclonal antibodies results in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models. Stimulation of VEGFR-3 augmented VEGF-induced angiogenesis and sustained angiogenesis even in the presence of VEGFR-2 (also known as KDR or FLK-1) inhibitors, whereas antibodies against VEGFR-3 and VEGFR-2 in combination resulted in additive inhibition of angiogenesis and tumour growth. Furthermore, genetic or pharmacological disruption of the Notch signalling pathway led to widespread endothelial VEGFR-3 expression and excessive sprouting, which was inhibited by blocking VEGFR-3 signals. Our results implicate VEGFR-3 as a regulator of vascular network formation. Targeting VEGFR-3 may provide additional efficacy for anti-angiogenic therapies, especially towards vessels that are resistant to VEGF or VEGFR-2 inhibitors.

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2+/−;Vegfr3+/− compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.

The Schlemm’s canal is a VEGF-C/VEGFR-3–responsive lymphatic-like vessel

In glaucoma, aqueous outflow into the Schlemms canal (SC) is obstructed. Despite striking structural and functional similarities with the lymphatic vascular system, it is unknown whether the SC is a blood or lymphatic vessel. Here, we demonstrated the expression of lymphatic endothelial cell markers by the SC in murine and zebrafish models as well as in human eye tissue. The initial stages of SC development involved induction of the transcription factor PROX1 and the lymphangiogenic receptor tyrosine kinase VEGFR-3 in venous endothelial cells in postnatal mice. Using gene deletion and function-blocking antibodies in mice, we determined that the lymphangiogenic growth factor VEGF-C and its receptor, VEGFR-3, are essential for SC development. Delivery of VEGF-C into the adult eye resulted in sprouting, proliferation, and growth of SC endothelial cells, whereas VEGF-A obliterated the aqueous outflow system. Furthermore, a single injection of recombinant VEGF-C induced SC growth and was associated with trend toward a sustained decrease in intraocular pressure in adult mice. These results reveal the evolutionary conservation of the lymphatic-like phenotype of the SC, implicate VEGF-C and VEGFR-3 as critical regulators of SC lymphangiogenesis, and provide a basis for further studies on therapeutic manipulation of the SC with VEGF-C in glaucoma treatment.

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfcum18 could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

VEGFR3 does not sustain retinal angiogenesis without VEGFR2

Significance VEGF/VEGFR and Notch signaling pathways are major molecular regulators of sprouting angiogenesis. Here we investigated the cross-talk between VEGFR and Notch signaling by using state-of-the-art inducible genetic mouse models. We found that VEGFR2 is absolutely required for the sprouting of endothelial cells that display low Notch activity. VEGFR3 cannot rescue the angiogenic sprouting associated with loss of Notch signaling. On the other hand, postnatal lymphangiogenesis required VEGFR3, but not VEGFR2. Because several new VEGF/VEGFR and Notch modulators are in clinical trials for the treatment of angiogenesis-related diseases, it is of the utmost importance to understand the signaling relationships of VEGFRs. This should also facilitate the design of more effective multitargeted treatments. Angiogenesis, the formation of new blood vessels, is regulated by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). VEGFR2 is abundant in the tip cells of angiogenic sprouts, where VEGF/VEGFR2 functions upstream of the delta-like ligand 4 (DLL4)/Notch signal transduction pathway. VEGFR3 is expressed in all endothelia and is indispensable for angiogenesis during early embryonic development. In adults, VEGFR3 is expressed in angiogenic blood vessels and some fenestrated endothelia. VEGFR3 is abundant in endothelial tip cells, where it activates Notch signaling, facilitating the conversion of tip cells to stalk cells during the stabilization of vascular branches. Subsequently, Notch activation suppresses VEGFR3 expression in a negative feedback loop. Here we used conditional deletions and a Notch pathway inhibitor to investigate the cross-talk between VEGFR2, VEGFR3, and Notch in vivo. We show that postnatal angiogenesis requires VEGFR2 signaling also in the absence of Notch or VEGFR3, and that even small amounts of VEGFR2 are able to sustain angiogenesis to some extent. We found that VEGFR2 is required independently of VEGFR3 for endothelial DLL4 up-regulation and angiogenic sprouting, and for VEGFR3 functions in angiogenesis. In contrast, VEGFR2 deletion had no effect, whereas VEGFR3 was essential for postnatal lymphangiogenesis, and even for lymphatic vessel maintenance in adult skin. Knowledge of these interactions and the signaling functions of VEGFRs in blood vessels and lymphatic vessels is essential for the therapeutic manipulation of the vascular system, especially when considering multitargeted antiangiogenic treatments.

VEGF‐C is required for intestinal lymphatic vessel maintenance and lipid absorption

Vascular endothelial growth factor C (VEGF‐C) binding to its tyrosine kinase receptor VEGFR‐3 drives lymphatic vessel growth during development and in pathological processes. Although the VEGF‐C/VEGFR‐3 pathway provides a target for treatment of cancer and lymphedema, the physiological functions of VEGF‐C in adult vasculature are unknown. We show here that VEGF‐C is necessary for perinatal lymphangiogenesis, but required for adult lymphatic vessel maintenance only in the intestine. Following Vegfc gene deletion in adult mice, the intestinal lymphatic vessels, including the lacteal vessels, underwent gradual atrophy, which was aggravated when also Vegfd was deleted. VEGF‐C was expressed by a subset of smooth muscle cells adjacent to the lacteals in the villus and in the intestinal wall. The Vegfc‐deleted mice showed defective lipid absorption and increased fecal excretion of dietary cholesterol and fatty acids. When fed a high‐fat diet, the Vegfc‐deficient mice were resistant to obesity and had improved glucose metabolism. Our findings indicate that the lymphangiogenic growth factors provide trophic and dynamic regulation of the intestinal lymphatic vasculature, which could be especially important in the dietary regulation of adiposity and cholesterol metabolism.

Tie1 deletion inhibits tumor growth and improves angiopoietin antagonist therapy

The endothelial Tie1 receptor is ligand-less, but interacts with the Tie2 receptor for angiopoietins (Angpt). Angpt2 is expressed in tumor blood vessels, and its blockade inhibits tumor angiogenesis. Here we found that Tie1 deletion from the endothelium of adult mice inhibits tumor angiogenesis and growth by decreasing endothelial cell survival in tumor vessels, without affecting normal vasculature. Treatment with VEGF or VEGFR-2 blocking antibodies similarly reduced tumor angiogenesis and growth; however, no additive inhibition was obtained by targeting both Tie1 and VEGF/VEGFR-2. In contrast, treatment of Tie1-deficient mice with a soluble form of the extracellular domain of Tie2, which blocks Angpt activity, resulted in additive inhibition of tumor growth. Notably, Tie1 deletion decreased sprouting angiogenesis and increased Notch pathway activity in the postnatal retinal vasculature, while pharmacological Notch suppression in the absence of Tie1 promoted retinal hypervasularization. Moreover, substantial additive inhibition of the retinal vascular front migration was observed when Angpt2 blocking antibodies were administered to Tie1-deficient pups. Thus, Tie1 regulates tumor angiogenesis, postnatal sprouting angiogenesis, and endothelial cell survival, which are controlled by VEGF, Angpt, and Notch signals. Our results suggest that targeting Tie1 in combination with Angpt/Tie2 has the potential to improve antiangiogenic therapy.

The Basis for the Distinct Biological Activities of Vascular Endothelial Growth Factor Receptor–1 Ligands

Differences in how two ligands interact with VEGFR-1 explain why only one ligand is strongly angiogenic. Defining Angiogenic Ability Angiogenic therapies could be useful in minimizing tissue damage caused by insufficiency of oxygen, whereas antiangiogenic therapies could help limit the growth of solid tumors. Binding of placenta growth factor (PlGF) to vascular endothelial growth factor receptor–1 (VEGFR-1) triggers angiogenesis in many tissues; however, VEGF-B, which also binds to VEGFR-1 with high affinity, is only weakly angiogenic. Anisimov et al. found that the L1 loop in PlGF, but not the corresponding loop in VEGF-B, interacted with a particular domain of VEGFR-1. Swapping the L1 loop between VEGF-B and PlGF showed that this domain was responsible for the differences in the ability of these ligands to activate VEGFR-1 and promote angiogenesis. These results may aid in designing VEGFR-1–targeting compounds that can inhibit or stimulate angiogenesis. Vascular endothelial growth factors (VEGFs) regulate blood and lymphatic vessel development through VEGF receptors (VEGFRs). The VEGFR immunoglobulin homology domain 2 (D2) is critical for ligand binding, and D3 provides additional interaction sites. VEGF-B and placenta growth factor (PlGF) bind to VEGFR-1 with high affinity, but only PlGF is angiogenic in most tissues. We show that VEGF-B, unlike other VEGFs, did not require D3 interactions for high-affinity binding. VEGF-B with a PlGF-derived L1 loop (B-L1P) stimulated VEGFR-1 activity, whereas PlGF with a VEGF-B–derived L1 loop (P-L1B) did not. Unlike P-L1B and VEGF-B, B-L1P and PlGF were also angiogenic in mouse skeletal muscle. Furthermore, B-L1P also bound to VEGFR-2 and activated downstream signaling. These results establish a role for L1-mediated D3 interactions in VEGFR activation in endothelial cells and indicate that VEGF-B is a high-affinity VEGFR-1 ligand that, unlike PlGF, cannot efficiently induce signaling downstream of VEGFR-1.

Comparison of vascular growth factors in the murine brain reveals placenta growth factor as prime candidate for CNS revascularization.

Vascular bypass procedures in the central nervous system (CNS) remain technically challenging, hindered by complications and often failing to prevent adverse outcome such as stroke. Thus, there is an unmet clinical need for a safe and effective CNS revascularization. Vascular endothelial growth factors (VEGFs) are promising candidates for revascularization; however, their effects appear to be tissue-specific and their potential in the CNS has not been fully explored. To test growth factors for angiogenesis in the CNS, we characterized the effects of endothelium-specific growth factors on the brain vasculature and parenchyma. Recombinant adeno-associated virus (AAV) vectors encoding the growth factors were injected transcranially to the frontoparietal cerebrum of mice. Angiogenesis, mural cell investment, leukocyte recruitment, vascular permeability, reactive gliosis and neuronal patterning were evaluated by 3-dimensional immunofluorescence, electron microscopy, optical projection tomography, and magnetic resonance imaging. Placenta growth factor (PlGF) stimulated robust angiogenesis and arteriogenesis without significant side effects, whereas VEGF and VEGF-C incited growth of aberrant vessels, severe edema, and inflammation. VEGF-B, angiopoietin-1, angiopoietin-2, and a VEGF/angiopoietin-1 chimera had minimal effects on the brain vessels or parenchyma. Of the growth factors tested, PlGF emerged as the most efficient and safe angiogenic factor, hence making it a candidate for therapeutic CNS revascularization.

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.