Marja Lohela

VEGFs and receptors involved in angiogenesis versus lymphangiogenesis.

Vascular endothelial growth factors and their endothelial tyrosine kinase receptors are central regulators of vasculogenesis, angiogenesis and lymphangiogenesis. VEGF signalling through VEGFR-2 is the major angiogenic pathway, and blockage of VEGF/VEGFR-2 signalling is the first anti-angiogenic strategy for cancer therapy. VEGFR-1 seems to act as a negative regulator of VEGF-mediated angiogenesis during development, and as a stimulator of pathological angiogenesis when activated by its specific ligands PlGF and VEGF-B. PlGF recruits angiogenic macrophages to tumours, and targeting PlGF could therefore be beneficial in cancer. For VEGF-B, with very limited angiogenic potential, a new role has been identified in regulating lipid metabolism in the heart. VEGF-C and VEGF-D induce lymphangiogenesis via VEGFR-3 and have also been shown to be lymphangiogenic in tumours, stimulating metastasis. Mouse models of lymphoedema have established VEGF-C as a promising agent for pro-lymphangiogenic therapy. In addition to lymphangiogenesis, VEGFR-3 has also been shown to be important for angiogenesis, acting together with VEGF/VEGFR-2 and Dll4/Notch signalling to control angiogenic sprouting. Increasing knowledge of the mechanisms regulating (lymph)angiogenesis should enable the development of better agents to combat metastasis and the resistance of tumours towards anti-angiogenic treatment, and of pro-(lymph)angiogenic treatment methods for ischaemic diseases and lymphoedema.

Lymphangiogenic growth factors, receptors and therapies

The lymphatic vasculature is essential for the maintenance of normal fluid balance and for the immune responses, but it is also involved in a variety of diseases. Hypoplasia or dysfunction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia or abnormal growth of these vessels are associated with lymphangiomas and lymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemic metastasis of cancer cells. Recent novel findings on the molecular mechanisms involved in lymphatic vessel development and regulation allow the modulation of the lymphangiogenic process and specific targeting of the lymphatic endothelium. Recent results show that the homeodomain transcription factor Prox-1 is an important lymphatic endothelial cell (LEC) fate-determining factor which can induce LEC-specific gene transcription even in blood vascular endothelial cells (BECs). This suggests that the distinct phenotypes of cells in the adult vascular endothelium are plastic and sensitive to transcriptional reprogramming, which might be useful for future therapeutic applications involving endothelial cells. Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are peptide growth factors capable of inducing the growth of new lymphatic vessels in vivo in a process called lymphangiogenesis. They belong to the larger family which also includes VEGF, placenta growth factor (PlGF) and VEGF-B, VEGF-C and VEGF-D are ligands for the endothelial cell specific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells which line the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosine kinase leads to primary lymphedema due to defective lymphatic drainage in the limbs. Other factors that seem to be involved in lymphangiogenesis include the Tie/angiopoietin system, neuropilin-2 and integrin alpha 9. VEGF-C induces lymphatic vessel growth, but high levels of VEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutant form of VEGF-C called VEGF-C156S lacks blood vascular side effects but is sufficient for therapeutic lymphangiogenesis in a mouse model of lymphedema. As VEGF-C156S is a specific lymphatic endothelial growth factor in the skin, it provides an attractive molecule for pro-lymphangiogenic therapy.

Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function.

Vascular endothelial cells are characterized by a high degree of functional and phenotypic plasticity, which is controlled both by their pericellular microenvironment and their intracellular gene expression programs. To gain further insight into the mechanisms regulating the endothelial cell phenotype, we have compared the responses of lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs) to vascular endothelial growth factors (VEGFs). VEGFR‐3‐specific signals are sufficient for LEC but not BEC proliferation, as shown by the ability of the specific ligand VEGF‐C156S to stimulate cell cycle entry only in LECs. On the other hand, we found that VEGFR‐3 stimulation did not induce LEC cell shape changes typical of VEGFR‐2‐stimulated LECs, indicating receptor‐specific differences in the cytoskeletal responses. Genes induced via VEGFR‐2 also differed between BECs and LECs: angiopoietin‐2 (Ang‐2) was induced via VEGFR‐2 in BECs and LECs, but the smooth muscle cell (SMC) chemoattractant BMP‐2 was induced only in BECs. Both BECs and LECs were able to promote SMC chemotaxis, but contact with SMCs led to down‐regulation of VEGFR‐3 expression in BECs in a 3‐dimensional coculture system. This was consistent with the finding that VEGFR‐3 is down‐regulated in vivo at sites of endothelial cell‐pericyte/smooth muscle cell contacts. Collectively, these data show intrinsic cell‐specific differences of BEC and LEC responses to VEGFs and identify a pericellular regulatory mechanism for VEGFR‐3 down‐regulation in endothelial cells.—Veikkola, T., Lohela, M., Ikenberg, K., Mäkinen, T., Korff, T., Saaristo, A., Jeltsch, M., Augustin, H. G., Alitalo, K. Intrinsic versus microenvironmental regulation of lymphatic endothelial cell phenotype and function. FASEB J. 17, 2006–2013 (2003)

Transgenic Induction of Vascular Endothelial Growth Factor-C Is Strongly Angiogenic in Mouse Embryos but Leads to Persistent Lymphatic Hyperplasia in Adult Tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.

Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 (Ang2) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell-cell junctions that form during lymphatic development.

Tie1 controls angiopoietin function in vascular remodeling and inflammation

The angiopoietin/Tie (ANG/Tie) receptor system controls developmental and tumor angiogenesis, inflammatory vascular remodeling, and vessel leakage. ANG1 is a Tie2 agonist that promotes vascular stabilization in inflammation and sepsis, whereas ANG2 is a context-dependent Tie2 agonist or antagonist. A limited understanding of ANG signaling mechanisms and the orphan receptor Tie1 has hindered development of ANG/Tie-targeted therapeutics. Here, we determined that both ANG1 and ANG2 binding to Tie2 increases Tie1-Tie2 interactions in a β1 integrin-dependent manner and that Tie1 regulates ANG-induced Tie2 trafficking in endothelial cells. Endothelial Tie1 was essential for the agonist activity of ANG1 and autocrine ANG2. Deletion of endothelial Tie1 in mice reduced Tie2 phosphorylation and downstream Akt activation, increased FOXO1 nuclear localization and transcriptional activation, and prevented ANG1- and ANG2-induced capillary-to-venous remodeling. However, in acute endotoxemia, the Tie1 ectodomain that is responsible for interaction with Tie2 was rapidly cleaved, ANG1 agonist activity was decreased, and autocrine ANG2 agonist activity was lost, which led to suppression of Tie2 signaling. Tie1 cleavage also occurred in patients with hantavirus infection. These results support a model in which Tie1 directly interacts with Tie2 to promote ANG-induced vascular responses under noninflammatory conditions, whereas in inflammation, Tie1 cleavage contributes to loss of ANG2 agonist activity and vascular stability.

Fighting vessel dysmorphia to improve glioma chemotherapy

High‐grade gliomas are aggressive and abundantly vascular tumors, and as in most cancer types, blood vessels in advanced lesions are highly abnormal. Poor perfusion and vascular leakage in tumor tissue resulting in hypoxia, necrosis, and high interstitial fluid pressure can hamper the efficient delivery of chemotherapy. Tumor angiogenesis is known to be supported by host leukocytes recruited to the tumor microenvironment, but the mechanisms leading to dysfunctional vascular network formation are incompletely understood. In this issue of EMBO Molecular Medicine, Mathivet et al ( ) present an elegant study, where longitudinal intravital imaging gives new insight on how recruitment and polarization of tumor‐associated macrophages regulate aberrant angiogenesis in experimental gliomas. They show that macrophage targeting results in vessel normalization and improved chemotherapy response, suggesting that the combination of these therapeutic modalities could improve the outcome of glioma treatment in the clinic.

Molecular Players in Lymphangiogenesis

Lymph vessels are essential for the maintenance of tissue fluid homeostasis and immune surveillance and are involved in the pathogenesis of several human diseases, such as lymphoedema, inflammation and tumour metastasis. The identification of lymphangiogenic growth factors and their receptor, and of transcription factors that control lymphangiogenesis, have provided significant insight into the biology of lymphatic vasculature. Another important advance has been the recent development of mouse, frog and fish models that allow the study of lymphangiogenesis and diseases affecting lymph vessels. In this review we discuss some of the major molecular players involved in regulation of lymphangiogenesis and lymph vessel remodelling, and the role of lymph vessels and their regulators in human disease.