Adult lymphangiogenesis is mostly quiescent, but its activity is obvious during tissue remodeling, inflammation, and tumor metastasis [178-180]. In fact, active lymphangiogenesis is essential for any organ transplantation. Organ transplantations thus provide evidence that adult lymphangiogenesis can be as massive as angiogenesis [181, 182]. At the cellular level, lymphangiogenesis is a complex process consisting of participation of multiple cell types and the release of lymphatic growth factors [183, 184].
Figure 5 provides a schematic illustration of lymphangiogenesis triggered by inflammation.
Sprouting of LVs from preexisting vessels is coordinated by macrophages, which secrete pro-lymphangiogenic signal activating LECs or macrophages may transdifferentiate into LECs [180]. Importantly, the adequate lymphangiogenic response requires a balance of pro- lymphangiogenic and anti-lymphangiogenic factors, otherwise lymphangiogenesis may be extensive but unproductive (creating nonfunctional LVs) [185]. One of the strongest pro- lymphangiogenic inducers are VEGFs, especially VEGF-C, as described in detail in the next chapter.
38 Figure 5: Schematic illustration of lymphangiogenesis during inflammation. Inflammatory stimulation through macrophage infiltration leads to sprouting of LVs from preexisting LVs. During this process, the VEGF-C/VEGFR-3 and VEGF-A/VEGFR-2 signaling pathways are activated.
Adapted from [180] and partially modified. Vascular endothelial growth factor receptor (VEGFR).
2.4.1 VEGF factors
There are five different growth factors in humans that belong to the VEGF family. VEGF- A, VEGF-B, VEGF-C, VEGF-D, and PGF. All members of the VEGF family are characterized by the central VEGF homology domain. Their function is based on respective receptors (i.e., VEGFR-2, VEGFR3) that possess cell-restricted expression [186]. VEGFR- 2 is expressed on LECs and BECs, respectively. During early development, VEGFR-3 is expressed in all endothelia but is later restricted to LECs and certain blood vascular ECs [186-188]. VEGF-A is described as a homodimeric glycoprotein with 9 expressed isoforms.
Isoform VEGF-A165 is physiologically most abundant [189, 190]. VEGF-A is regarded as the strongest pro-angiogenic activator involved in angiogenesis under physiological and pathological conditions [191, 192]. VEGF-B is another member of VEGF family with poor angiogenic role in most tissue. Deletion of VEGF-B in heart-mouse model resulted in changes of atrial conduction characterized by a prolonged PQ interval [193] but on the other hand overexpression is associated with cardiomyocyte hypertrophy in transgenic mouse model [194]. VEGF-C and VEGF-D are next two important VEGF members described more extensively in recent years. VEGF-D is a VEGFR-3 ligand that is capable of stimulating
39 lymphangiogenesis during overexpression, but its loss in VEGF-D deficient mice does not lead to an obvious phenotype [195]. Maybe this is one of the reasons why researchers have focused more intensively on VEGF-C research observations.
2.4.2 VEGF-C
VEGF-C is considered to be the main lymphangiogenic factor, as confirmed by a number of research studies [186, 196-198]. VEGF-C was discovered more than 20 years ago. Joukov et al. [187] showed that the isolated cDNA encodes a protein that is proteolytically processed, secreted into the cell culture medium and binds to both extracellular domains of VEGFR-3 and VEGFR-2 thereby triggering the induction of tyrosine autophosphorylation.
Later, it was observed that VEGF-C stimulates ECs migration in collagen gels. Jeltsch et al.
[196] have demonstrated that overexpression of VEGF-C in the skin of transgenic mice results in lymphatic proliferation. Moreover, VEGF-C induces the permeability of blood vessels, leading to an angiogenic effect in prenatal development [199]. In the coculture model, LECs, BECs and ASCs in a 3D fibrin matrix depended on the addition of exogenous VEGF-C to form a typical LEC's network [200].
2.4.2.1 Biosynthesis and activation
VEGF-C is produced primarily in the large intestine and the mammary duct epithelium, as well as in skeletal and cardiac muscle, where lymphangiogenesis is enhanced by ICs [197].
As depicted in Figure 6 [186] the protein is produced as an inactive prepropeptide consisting of silk homology domain, VEGF homology domain, N-erminal propeptide domain, and signal domain. Proprotein convertases furin, PC5, and PC7 cleave intracellularly the prepropeptide between the VEGF homology domain and the C-terminal domain, resulting in still inactive VEGF-C (pro-VEGF-C). The silk homology of two inactive VEGF-C molecules are covalently connected via cysteine bridges to form the dimer form of pro- VEGF-C. Then, pro-VEGF-C is secreted from cells. It was shown that pro-VEGF-C is capable of binding to VEGFR-3, but it does not trigger the phosphorylation of the receptor.
Thus, a second, extracellular, proteolytic cleavage is necessary to produce the active form of VEGF-C. During embryonic development, ADAM Metallopeptidase with Thrombospondin Type 1 Motif (ADAMTS) 3 results in the removal of both terminal domains of pro-VEGF-
40 C and generates major form of mature active VEGF-C. The minor form is likely a product of plasmin cleavage that is about nine amino acids longer compared to the major form of VEGF-C [186]. In addition, ADAMTS3-mediated cleavage of VEGF-C was more efficient in the presence of Collagen and Calcium Binding EGF Domains 1 (CCBE1) [201, 202].
Fully processed VEGF-C is then suitable for activation of the VEGF-C/VEGFR-3 signaling pathway.
Figure 6: Schematic illustration of biological activation of VEGF-C. Adapted from [186].
Furthermore, as previously mentioned, the ability of ADAMTS3 to cleave pro-VEGF-C to active VEGF-C has been clearly established during embryonic life. However, proteolytic activation must also occur in adulthood to ensure an adequate response to lymphatic homeostasis. However, as ADAMTS3 has a very low expression in adulthood, additional protease must be involved in VEGF-C maturation. High sequence homology and identical domain composition analyses have identified that ADAMTS2 and ADAMTS14 are suitable for pro-VEGF-C mediated cleavage and Dupont et al. [203] demonstrated that ADAMTS2 and ADAMTS14 are capable of processing pro-VEGF-C into active VEGF-C as efficiently as ADAMTS3. Importantly, based on the snRNAseq data, ADAMTS2 is also expressed by cells of mesenchymal origin, including fibroblasts and adipocytes [2]. Thus, AT could be the site of VEGF-C activation, but this has not been experimentally proven yet. Therefore, part of my thesis was dedicated to experiments designed to test the hypothesis that
41 adipocytes may affect lymphangiogenesis by providing factors necessary for VEGF-C activation.
2.4.2.2 Molecular regulation: VEGF-C/VEGFR-3 signaling
As mentioned above, VEGF-C can activate VEGFR-3 expressed by LECs. The immunoglobulin-like domains of VEGFR-3 are responsible for ligand binding, while the domains closest to the membrane are important for receptor homodimerization. VEGFR-3 can form homodimers or heterodimers with the VEGFR-2 receptor, which transduces signals through the Ras/Ras/MEK/ERK pathway. Signal transduction is characterized by the phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (AKT) pathway. By the time AKT is phosphorylated, activation of mammalian targets of rapamycin and Rac 1 is accomplished [1]. Activation of these signaling pathways is a prerequisite of LECs lymphangiogenesis as described by Figure 7 [1]. Morfoisse et al. [204] have shown that apart from VEGF-C, lymphangiogenesis requires fuel in the form of FFAs, namely oleic acid.
Figure 7: Schematic illustration of the VEGF-C/VEGFR-3 signaling pathway. Adapted from [1] and later modified.
42