REVIEW ARTICLES

The Proteolytic Activation of Vascular Endothelial Growth Factor­C

M. Lackner1, C. Schmotz2, M. Jeltsch3,4 1 University of Hamburg, Germany; 2 Research Program for Clinical and Molecular Metabolism, University of Helsinki, Finland; 3 Individualized Drug Therapy Research Program, University of Helsinki, Finland; 4 Wihuri Research Institute, Helsinki, Finland

Summary Die proteolytische Aktivierung des Vas­ Aktivität von VEGF­C für verschiedene spe ­ The enzymatic cleavage of the protein kulären Endothelzellwachstumsfaktors­C zi fische Funktionen von unterschiedlichen back bone (proteolysis) is integral to many Pro teinasen regu liert wird. Obwohl VEGF­C biological processes, e.g. for the break­ Zusammenfassung in experimentellen Tierversuchen eine zen­ down of proteins in the digestive system. Enzymatische Schnitte der Polypeptidkette trale Stellung in der Tumorprogression und Specific proteolytic cleavages are also used von Proteinen sind Bestandteil vieler biolo­ ­metastasierung einnimmt, ist die Aus­ to turn on or off the activity of proteins. For gischer Prozesse, so z.B. bei der Zerlegung sagekraft der bisherigen korrelativen Stu­ example, the lymphangiogenic vascular von Proteinen während der Verdauung. dien zur Rolle von VEGF­C bei Tumor er ­ endothelial growth factor­C (VEGF­C) is Gezielte enzymatische Schnitte werden kran kungen des Menschen begrenzt. synthesized as a precursor molecule that auch benutzt, um die Aktivität bestimmter Dieser Umstand ist nicht zuletzt in den must be converted to a mature form by the Proteine ein­ oder auszuschalten. So z.B fehlenden Möglichkeiten begründet, zwis­ enzymatic removal of C­ and N­terminal wird der lymphangiogene Vaskuläre En­ chen der inaktiven und den aktiven For­ pro peptides before it can bind and activate dothelzellwachstumsfaktor­C (VEGF­C) als men zu unterscheiden. its receptors. The constitutive C­terminal Vorläufermolekül synthetisiert, welches Schlüsselwörter: VEGF­C, Lymphangio­ cleavage is mediated by proprotein con­ durch die enzymatische Entfernung von genese, Proteinasen, Proteolyse vertases such as furin. The subsequent ac­ Teilen des Proteins in eine aktive Form tivating cleavage can be mediated by at umgewandelt werden muss, bevor es seine least four different proteases: by plasmin, Rezeptoren binden und aktivieren kann. ADAMTS3, prostate­specific antigen (PSA) Dabei werden C­ und N­terminale Propep­ List of Abbreviations and cathepsin D. Processing by different tide von VEGF­C entfernt. Die konstitutive proteases results in distinct forms of "ma­ C­terminale Spaltung wird durch Propro­ ADAM A Disintegrin­ and Metalloproteinase ture" VEGF­C, that differ in their affinity and teinkonvertasen wie Furin vermittelt. Die their receptor activation potential. This darauffolgende, aktivierende Spaltung ADAMTS3 A Disintegrin­ and Metalloproteinase processing is tightly regulated by the kann durch mindestens vier verschiedene with Thrombospondin Motifs­3 CCBE1 protein. CCBE1 regulates the acti­ Proteasen vermittelt werden: Plasmin, CatD Cathepsin D vating cleavage of VEGF­C by ADAMTS3 ADAMTS3, prostata­spezifisches Antigen and PSA, but not by plasmin. During em­ (PSA) und Cathepsin D. Resultierend aus CCBE1 Collagen­ and Calcium­Binding EGF bryonic development of the lymphatic sys­ der Prozessierung durch unterschiedliche domain­containg protein 1 tem, VEGF­C is activated primarily by the Proteasen entstehen unterschiedliche, ADAMTS3 protease. In contrast, it is be­ “reife” VEGF­C­Formen, die sich in ihrer Affi ­ ELISA Enzyme­Linked Immunosorbent Assay lieved that plasmin is responsible for ni tät und ihrem Rezeptor­Akti vier ungs po­ wound healing lymphangiogenesis and tential unterscheiden. Das CCBE1­Protein ECM Extracellular Matrix PSA for tumor­associated pathological lym­ reguliert die Prozessierung von VEGF­C phangiogenesis. Cathepsin D has also been durch ADAMTS3 und PSA, aber nicht die HS Hennekam Syndrome implicated in tumor lymphangiogenesis. In durch Plasmin. Während des physiologis­ addition, cathepsin D in saliva might acti­ chen Wachstums des Lymphgefäßsystems HSPG Heparansulfate Proteoglykan vate latent VEGF­C upon wound licking, in der Embryonalentwicklung wird VEGF­C there by accelerating wound healing. The hauptsächlich durch die ADAMTS3­Pro­ KLK3 Kallikrein­related Peptidase 3 molecular details of proteolytic activation tease aktiviert. Im Unterschied dazu wird (synonymous with PSA) of VEGF­C are only recently extensively ex­ vermutet, dass Plasmin für die Wund­ plored, and we likely do not know yet all heilung und PSA für die mit Tumorwachs­ MMP­3 Matrix­Metalloproteinase­3 ac ti vating proteases. It appears that the ac­ tum assoziierte, pathologische Lymph an ­ tivity of VEGF­C is regulated for different gio genese verantwortlich ist. Cathepsin D PSA Prostata­Specific Antigen spe cific functions by different proteinases. wurde ebenfalls mit der Tumor­Lymphan­ Al though VEGF­C clearly plays a pivotal giogenese in Verbindung gebracht; da rü ­ TGF­β Transforming Growth Factor­β role for tumor progression and metastasis ber hinaus könnte das im Speichel ent hal ­ in experimental animal studies, the rele­ tene Cathepsin D, durch Wundlecken, la ­ VEGF­A Vascular Endothelial Growth Factor­A, vance of most correlative studies on the ten tes VEGF­C aktivieren und dadurch die often simply referred to as “VEGF” role of VEGF­C in human cancers is quite li ­ Wund heilung beschleunigen. Die moleku­ mi ted until now, also due to the lack of laren Details der proteolytischen Akti vier ­ VEGF­C, ­D Vascular Endothelial Growth Factor­C, ­D meth ods to differentiate between inactive ung von VEGF­C werden erst seit kurzer and active forms. Zeit intensiv erforscht und wahrscheinlich VEGFR VEGF Receptor Key Words: VEGF­C, lymphangiogenesis, sind noch nicht alle aktivierenden Prote a ­ proteinases, proteolysis sen bekannt. Jedoch scheint es, dass die VHD VEGF Homology Domain

88 LymphForsch 23 (2) 2019 REVIEW ARTICLES

Proteinases (protein generated from the prec ursor trypsi - cleaving enzymes) no gen by auto catalysis.

Proteinases (or pro te ases) are enzymes that cleave pro - Activation of proteins teins by hydrolysing the peptide bonds of the protein Many pro teins are produced as backbone (Figure 1). ey oc - inactive pre cur sors and are activated cur in side (intracellular) and by proteolytic cleavage when their out side (extracellular) of cells, function is required. e proteinases and are es sential for a multi - them selves are also produced as in - tude of cell and body func- active pro-proteinases and must be ac - tions. For example, proteinases ti vated by pro teolytic removal of their process antigens in the course pro peptides. is is of uppermost im - of an im mune reaction for por tance, since the uncon trolled acti - antigen presentation, they vi ty of proteinases would other wise de - break down damaged or un ne - stroy cells and decompose the extra cel - Figure 1 ces sary proteins (e.g. in lyso - lular matrix (ECM). Proteolysis. The hydrolytic cleavage of a peptide bond somes) and they digest food e perhaps best known pro teo ly ti - (orange) of a protein (blue background) into two proteins in the gastro in tes tinal cal ly con trolled processes include fragments (red background). The peptide bonds of the tract. In the stomach, for blood coagu la tion, the limitation of protein backbone are shown as thick lines. The amino example, pepsin is generated blood clotting and its reversal, i.e. the acid side chains are symbolized as green circles. Without from the precursor pepsinogen dis solution of blood clots [1,2]. Many enzymatic catalysis by proteinases this chemical by autoproteolysis at a low pH, bood clotting factors are proteinases, reaction is extremely slow. and in the intestine, trypsin is which in turn acti vate other pro te in a - ses, etc. ("proteolytic cas cade") to cata - lyze the proteolytic con version of so lu - ble fibrinogen into poly merizing fibrin in the final step of the blot clotting cas - cade.

Activation of growth factors

Many growth factors and also some cyto kines are produced as inactive pre - cur sors, which only become active through pro teo lytic cleavage (“pro ces - sing”). Among the better known growth factors that are activated through proteolytic cleavage are e.g. the Transforming Growth Factor-β (TGF-β)[3], but also the lymph an gio - ge nic growth factors VEGF-C and VEGF-D. Many studies have analyzed the me cha nisms and the regulation of Figure 2 VEGF re cep tor activation by VEGFs The growth factors VEGF-A, VEGF-C and VEGF-D and their receptors. The growth and [4], whereas rela tive ly little is known function of blood and lymphatic vessels is controlled by Vascular Endothelial Growth Factors about the upstream pro cesses of mo bi - (VEGFs). VEGF­A is the quintessential growth factor for blood vessels, while VEGF­C is the li sation and activation of VEGFs. quintessential growth factor for lymphatic vessels. VEGF­A is recognised by VEGF receptor­1 (VEGFR­ 1) and VEGF receptor­2 (VEGFR­2). VEGF­C and VEGF­D are recognised by VEGF receptor­3 (VEGFR­3) and, under certain circumstances, also by VEGFR­2. VEGFR­1 is largely specific for endothelial cells of The VEGF family blood vessels and VEGFR­3 for endothelial cells of lymphatic vessels. In contrast, VEGFR­2 is found on e biology of the growth factors both vessel types. If, for example, active VEGF­C or VEGF­D binds to VEGFR­3 on the lymphatic VEGF-C and VEGF-D has been de - endothelial cell surface, the signal is transduced into the cell nucleus, where it provokes a scribed in detail in a previous review proliferative and migratory response, thus initiating vessel growth.

LymphForsch 23 (2) 2019 89 REVIEW ARTICLES

The C-terminal domain of VEGF-C

Similar to VEGF-A, VEGF-C and VEGF-D are also im mo bi lized on cell sur faces and the ECM via their C-ter - minal domain [9]. In contrast to all other VEGF family members, the C- ter minal domain of VEGF-C and VEGF-D blocks the growth factor acti - vity [10]. Most likely, this do main ste - ri cally hinders access to the re cep tor bin ding site. is assumption would also explain why the C-terminal do - Figure 3 main of VEGF-C is almost twice as Schematic representation of the domain organisation of VEGF growth factors using VEGF- large as its VHD. e origin of the pro - C/D and VEGF-A as examples. The VEGF growth factors consist of the central VEGF homology tein sequence of the C-terminal do - domain (in grey) and optional accessory domains (in blue and magenta). The proteolytic cuts main is mysterious, since no homo lo - usually take place between the domains (in red). The characteristic cysteine patterns of the VEGF gous sequences seem to exist in the ge - family and the C­terminal propeptide are represented by yellow and white lines, respectively. nomes of verte brates. Homologous pro teins are, however, found in the sa - li vary secretions of some silk worm [5]. For this reason, only a short intro - which they bind heparan sulfate pro - mos quito larvae, e.g. Chi ro no mus duction follows, in which the relevant teo gycans. is variation in binding tentans [11]. For this reason, this do - pro perties and cha rac teristics of strength causes a more or less pro mi - main has also been called silk homo - VEGF-C and VEGF-D are explained. nent immobilization on cell sur faces logy do main, although its amino acid VEGF-C and VEGF-D belong to the and the extracellular matrix (ECM), se quence is unre lated to the classical VEGF family (see also Figure 2 for a which in turn results in distinct ac ti vity silk proteins. gra phical short overview of VEGF-A, pro files of the isoforms. Proteases such VEGF-C, VEGF-D and their re cep - as plasmin can convert the longer Hypoxia regulates angio- tors). ECM-bound VEGF-A iso forms into genesis, but how is lym- Charac teristic for the members of shor ter, more diffusible iso forms [6,7]. phangiogenesis regulated? the VEGF fa mily is the VEGF Homo - Cleavage by different matrix me tallo - logy Domain (VHD) as the central and pro teinases (MMPs), especially MMP- VEGF-A, which is mainly re spon si - do minant structural element. is 3, converts e.g. the main isoform ble for the for mation of blood vessels, domain is almost 100 amino acids long VEGF-A165 into a shorter, non-heparan is tightly regulated at the trans crip ti o - and has a characteristic arrangement of sul fate-binding isoform [8]. nal level. If the oxygen sup ply to a eight cysteine (C) amino acid residues

(CX22CPXCVX3RCXGCCX6CX33-35CXC), which form disulfide bridges among them selves and thereby give the VEGFs a very stable core. is core also forms the re cep tor binding epi - tope and thus determines to which of the three VEGF receptors (-1, -2 and -3) a VEGF binds. In addi tion to this core, most VEGFs have other domains that are either upstream (N-terminal) or downstream (C-ter mi nal) from the VHD (Figure 3). ese ad ditional do - mains give VEGFs the abi li ty to inter - Figure 4 act with other binding part ners. Schematic representation of known control loops in angiogenesis and For example, different isoforms of lymphangiogenesis. VEGF-A have C-terminal heparin bin - The production of VEGF­A and VEGF­C is usually self­limiting due to negative feedback. As soon as a ding domains of varying strength, with sufficient oxygen supply has been established or the tissue pressure normalized, the signaling for blood vessel or lymphatic growth is reduced.

90 LymphForsch 23 (2) 2019 REVIEW ARTICLES tissue is insufficient (hy po xia), the production of VEGF-A is switched on, which in turn leads to blood vessel growth and normalisation of the oxy - gen pressure [12]. In contrast, VEGF-C pro duction hardly im proves tissue oxy genation, but it does im prove tissue drai nage and immune cell trafficking (see Figure 4). Presumably for this rea - son and in contrast to VEGF-A, the pro duction of VEGF-C is controlled by pro inflammatory signals and not or only insignificantly by hypoxia [13-15]. In addition, VEGF-C can contribute to the limitation of inflammatory re - actions by in creased drainage [16,17] and im mu no modu lation [18]. VEGF- C also plays a vital role for the lymph ves sels of the small in tes tinal villi (lacteals). ese in fat absorption spe - cia lised vessels require for their main - ta nance the permanent stimulation by Figure 5 VEGF-C [19], which is produced by Schematic representation of the proteolytic activation of VEGF-C. VEGF­C is synthesized as a ma crophages in response to the mi cro - precursor with a size of 58 kDa. This unprocessed form (also called “prepro­VEGF­C”) is more than bial intestinal flora [20]. twice as large as the mature VEGF­C and, after the signal peptide has been cleaved off during Increased interstitial tissue pressure transport into the endoplasmic reticulum, is converted into pro­VEGF­C in the trans­Golgi network am pli fies the growth of lymph vessels by the proprotein convertases PC5, PC7 and especially furin. This occurs by cleaving the polypeptide via the pressure-dependent signal chain C­terminally to the VHD (marked by a yellow triangle). If furin is blocked, unprocessed VEGF­C trans duc tion of VEGF receptor-3 (me - is not converted into pro­VEGF­C [23]. Pro­VEGF­C can bind but not activate VEGFR­3 and therefore chano trans duction) mediated by β1 acts as a competitive inhibitor of active VEGF­C, which has been shown both in vitro and in vivo [24]. integrin and integrin-linked kinase Only a further proteolytic cut N­terminally of the VHD (marked by red triangles) converts pro­VEGF­C (ILK), there by normalising tissue pres - into the biologically active form. Mature VEGF­C has by far the highest affinity for the binding and sure [21,22]. Whether tissue pres sure activation of VEGFR ­2 and ­3 [10]. Unprocessed VEGF­C is hardly detectable in the cell culture also has an in fluence on VEGF-C pro - supernatant and probably occurs physiologically only inside the cell [10]. duc tion or activa tion is not known yet. throm bospondin type 1 motif. Some Although the biosynthesis of VEGF- fun ctions of this protein family, such as D is very similar to that of VEGF-C During embryonic develop- pro collagen processing or pro teo glycan (shown sche matically in Figure 5) [34], ment VEGF-C is activated by clea vage, have been linked to the regu - ADAMTS3 cannot activate VEGF-D ADAMTS3 la tion of angio gene sis [28]. Due to its [26,35]. And, notably, none of the struc ture, ADAMTS3 belongs to gether other procollagenases of the ADAMTS Mutations in the Collagen- and Cal - with ADAMTS2 and ADAMTS14 to family (ADAMTS2 and ADAMTS14) ci um-Binding EGF domain-containg the procollagenase group [29] and also do activate VEGF-C [26]. pro tein 1 (CCBE1) gene are respon si - cleaves, at least in vi tro, procollagen N- ble for the systemic lymphatic dys - pro peptide [30]. plasia in Hennekam Syndrome Type I Activation of VEGF-C in [25]. CCBE1 regulates the proteinase If the function of ADAMTS2 is mu - wound healing by plasmin ADAMTS3, which is the primary pro - ta tionally im paired, proteolytic col - and cathepsin D tein ase that activates VEGF-C du ring lagen maturation is disturbed and a em bryonic growth [26,27]. e con nective tissue defect is the con se - e restoration of oxygen supply and ADAMTS proteinases are cell surface quence (Ehlers-Danlos syndrome, der - im mune function through blood and or ECM-localized multidomain en - ma to sparaxis type) [31]. In contrast, lymph vessels are paramount for zymes close ly related to the ADAM pa tients without or with compromised wound healing. An acceleration of pro teinases. In contrast to the mem - ADAMTS3 genes show no deficits in wound healing by VEGF-C was first brane-bound ADAM proteinases, the col lagen synthesis, but distinct defects ob served in animal experiments in ADAMTS proteinases are secreted and in the development of the lymphatic 2004 [36,37]. When platelets are acti - con tain one or more repeats of the sys tem [32,33]. va ted, VEGF-C is released from the α-

LymphForsch 23 (2) 2019 91 REVIEW ARTICLES

bio logical task is to liquefy the gel-like con sistency of the male ejaculate, which allows the sperm cells to swim [46]. VEGF-A had been detected in semi - nal fluid more than 20 years ago [47,48], and was later shown to have a po si tive effect on the mo ti lity of sper - ma tozoa [49]. How ever, only recently it was recognized that also VEGF-C is pre sent in the male ejaculate and that the ac ti vation of this seminal VEGF-C oc curs concurrently with the li que fac - tion of the ejaculate by PSA [42]. Whether seminal VEGF-C is an epi - phe nomenon or has any function for re pro duction has not yet been clarified. VEGF-C is certainly required for the Figure 6 im plan tation of the embryo into the Schematic representation of the proteolytic processing sites in the amino acid sequences endo me trium, where it acts on the of VEGF-C and VEGF-D. The activation of VEGF­C and VEGF­D is achieved by proteolytic cleavage blood vessels [33]. However, VEGF-C of the protein between the N­terminal domain and the VEGF homology domain. The further C­ could also play a role in the implan ta - terminally (in the figure to the right) the cleavage of VEGF­C occurs, the lower the biological activity tion-associated immune modu la tion of the resulting VEGF­C form [42]. The cleavage of VEGF­C by plasmin within the VEGF homology [50] or it might - as already described domain leads to a complete inactivation of VEGF­C [26]. Alternatively, VEGF­C can also be for VEGF-A [49] - have a direct che - inactivated through sequesteration by soluble isoforms of its receptors [44]. Figure 6 was modified mo tactic or chemokinetic effect on under the Creative Commons license from [42]. sper matozoa. gra nules [38]. Plas min, which is later which is con troversially used in pro - in volved in the dis solution of the tem - state cancer screening, can acti vate The key position of CCBE1 as po rary fibrin ma trix [39], probably ac - VEGF-C [42]. Despite being the most cofactor of activation ti vates both plate let-derived VEGF-C fre quently used blood test for early and latent VEGF-C (pro-VEGF-C can cer detection, it is less well known When it is important to react quickly which was em bed ded in the extra cel lu - that PSA is a proteinase whose main to chang ing demands, regulation at the lar matrix [40, 41]). Cathepsin D is another activator of VEGF-C [42]. Ac celerated wound hea - ling by saliva [43] can per haps be part - ly attri bu ted to the acti va tion of VEGF-C by Cathepsin D, which is found in saliva. However, many other en zymes are re leased during wound heal ing which have a fairly broad sub - strate spe ci fi ci ty, e.g. MMP-3, and which may contribute to the acti va tion of VEGF-C as well as to the re lease/ac - ti vation of ECM-sequestered (in active) Figure 7 VEGF-A. Schematic representation of the hypothetical mechanism of action of CCBE1. The C­termi­ nal domain of pro­VEGF­C (dark blue) blocks the access of enzymes to proteolytically sensitive se­ Activation of VEGF-C by quences (shown on the left half of the dimer). CCBE1 causes a conformational change in VEGF­C prostate specific antigen and thus exposes the proteolytic target site (shown on the right half of the dimer). The CCBE1 effect (PSA, KLK3) on VEGF­C activation has been demonstrated for ADAMTS3 and KLK3/PSA , and it is suspected for Cathepsin D. After the activation of VEGF­C, e.g. at the primary interface of plasmin (#1), Cathepsin Somewhat surprisingly, but not en ti - D can shorten the protein with an additional cut (“secondary activation”). The shorter the N­termi­ re ly unanticipated [45], it turned out nal end of active VEGF­C, the weaker it binds to and activates its receptors. With a proteolytic cut at that PSA (pros tate specific anti gen), the secondary plasmin cleavage site (#2), VEGF­C loses all activity towards VEGFR­2 and VEGFR­3.

92 LymphForsch 23 (2) 2019 REVIEW ARTICLES gene expresion level introduces a delay due to the upstream pro cessess of trans cription and translation. e pro - duction and on-demand activation of in active ("latent") VEGF-C bypasses this delay. A si mi lar form of storage and activation is known e.g. from TGF-β [51]. e heparin-binding iso - forms of VEGF-A are also reversibly in activated by binding to extracellular pro teins and can be re ac ti vated if re - quired, e.g. by plasmin-mediated pro - teo lytic cleav age [6]. A summary of all pre viously published VEGF-C acti va - ting enzymes and the exact positions of the cleavage sites is shown in Figure 6. e CCBE1 protein re gulates the VEGF-C-activating func tion of the ADAMTS3 proteinase. CCBE1 con sists of two domains: the N-terminal do - main, which is formed by three EGF- Figure 8 like repreats, and the C-terminal do - The different activation paths of VEGF-C. The proteolytic cleavage of pro­VEGF­C by ADAMTS3 main. which consists of two collagen activates and mobilizes VEGF­C. The activation of VEGF­C can take place in four different settings: mo tifs. Both do mains are able to ac ce - 1. activation of VEGFR­3­bound pro­VEGF­C [26] le rate the acti va tion of VEGF-C by 2. activation of HSPG­bound VEGF­C [9] ADAMTS3 independently. e N-ter - 3. activation of VEGF­C in the soluble phase [35] mi nal domain of CCBE1 is res pon sible 4. activation of ECM­bound VEGF­C [40]. for the colocalization of VEGF-C and VEGFR­3­bound but inactive VEGF­C can start signaling immediately after proteolytic activation ADAMTS3 with CCBE1 to form the (activation mode 1), whereas HSPG­bound VEGF­C must first dissociate from the HSPG and ac ti vation complex, and the C-term i - translocate to VEGFR­3 (activation mode 2). The activation of VEGF­C can also take place in the nal do main accelerates the catalytic soluble phase (activation mode 3). Immunohistochemically, however, the vast majority of pro­ clea vage of VEGF-C by ADAMTS3 VEGF­C, CCBE1 and ADAMTS3 are found bound to the extracellular matrix (ECM, activation mode [40]. Pre su ma bly, CCBE1 removes the 4) or on cell surfaces (activation modes 1 and 2). CCBE1 fulfills two independent functions for mas king of the proteolytic target site of VEGF­C activation: the C­terminal domain accelerates the proteolytic cleavage, while the N­ VEGF-C, which is normally blocked terminal domain recruits pro­VEGF­C to efficiently form the trimeric activation complex. Figure 8 by its own C-terminal domain (Figure was modified under the Creative Commons license from [40]. 7). e different acti va tion paths of VEGF-C with regard to the localisation Hennekam Syndrome (HS) is a rare first, mutations in the CCBE1 gene of the activation com plex are explained con genital disease with a generalized were identified as the cause, but mean - in Figure 8. lymph edema as its main feature. At while, mutations in three different

Proteinase Cleavage site Remarks Activates the following receptors Reference(s)

VEGFR­2 Plasmin (primary cleavage site) Arg102↓Thr103 minor form, probably responsible for the activation of [26,41] VEGF­C in wound healing VEGFR­3

↓ main form, for lymphangiogenesis during embryonal VEGFR­2 ADAMTS3 Ala111 Ala112 development, CCBE1­ regulated VEGFR­3 [26,32,33,40]

Tyr114↓Asn115 VEGFR­2 KLK3/PSA activates VEGF­C in seminal fluid, CCBE1­ regulated VEGFR­3 [42]

Cathepsin D Leu119↓Lys120 found in seminal fluid and saliva preferentially VEGFR­3 [42]

Plasmin (secondary cleavage site) Arg127↓Lys128 inactive form [26]

Table 1 Four proteinases are known in the literature as VEGF-C activators. Plasmin occupies a special position because it inactivates VEGF­C during prolonged exposure by cutting at a secondary site.

LymphForsch 23 (2) 2019 93 REVIEW ARTICLES genes are known to trigger HS. e func tion of two of these genes (CCBE1 and ADAMTS3) within the VEGF-C sig nal transduction pathway is known. It is assumed that the third gene (FAT4) also has an important function with in the VEGF-C signal trans - duction path way.

Activation of VEGF-C in tumours

VEGF-C and its activation are in dis - pen sable for the development of the lym phatic system [33,52], and in the adult organism, at least some lym - phatic networks need a constant supply of VEGF-C for their maintenance [19]. To prevent lymphatic dysfunction, the amount of active VEGF-C must be Figure 9 Gene therapy with AdVEGF-C. An increasingly popular therapy for breast cancer­associated pre cise ly regulated. A degregulation lymphedema is autologous lymph node transplantation [88,89]. In pre­clinical studies, the with severe consequences can e.g. be treatment success (integration of the transplanted lymph node into the local lymphatic network) trig gered by tu mors. could be improved by the simultaneous administration of VEGF­C [90]. With this strategy, e relationship between VEGF-A- Lymfactin® has successfully completed Phase I clinical trials and is now in Phase II. As a further me di ated blood vessel formation and development of Lymfactin® , a simultaneous administration of VEGF­C with the VEGF­C­activating tu mour growth has been well studied ADAMTS3 and/or CCBE1 is being discussed. and is also spe ci fically blocked in anti- an gio genic tu mour therapy, e.g. by the press VEGF re cep tors and be stimu la - thep sin D, the corre la tion between anti body drug be va cizumab (Avastin) ted in an autocrine or paracrine PSA and tumour develop ment has [53]. It has always been assumed that fashion by VEGF-C [59]. been studied much more in ten sively, the majority of tumours never be come va rious studies have come to dif ferent cli ni cally relevant because they do not 4. VEGF-C can stimulate lymph ves - con clusions regarding a tu mour-pro - ac quire the ability to stimulate blood sel growth and thus promote metas - mo ting function of PSA [65-69]. Some ves sel growth [54]. Without switching tasis [60-62]. au thors postulate that PSA pro motes on VEGF-A production and without Unlike for the blockade of VEGF-A, early tumour growth but in hi bits its the re sul ting vascularisation ("angio- there is no approved drug therapy for de v elop ment in later stages [70]. In any ge nic switch"), these tumours can ne - the blo ck ade of VEGF-C. is lack case, with the activation of VEGF-C by ver grow lar ger than a few mil li metres might result from the fact that pro te o - ca thep sin D and PSA, pos si ble mecha - because they lack sufficient oxy gen and ly tic activation produces many dif fer - nis tic links have been iden ti fied, which nutrients [55,56]. ent forms of VEGF-C. Effective block - al lows to experimentally address and How ever, tumours can produce not ing would likely need to block all ans wer these and similar questions. only VEGF-A but also VEGF-C. e forms of VEGF-C in addition to all ef fects of VEGF-C on tumour growth forms of VEGF-D, as VEGF-D can oc cur at several levels: pro vide si mi lar signals for tumour Pro-VEGF-C or active VEGF-C? growth as VEGF-C [63]. 1. VEGF-C can activate VEGFR-2 e vast majority of studies on the Which proteinases activate VEGF-C and thus replace VEGF-A as an angio - role of VEGF-C in tumor growth de - in tu mour diseases has not yet been ge nic factor [57]. scribe the correlation of VEGF-C levels ex pe ri mentally investigated, but ca - 2. VEGF-C can stimulate VEGFR-3, with di sease progression. However, thep sin D and PSA are likely to play a which is found particularly on newly none of these studies distinguishes bet - role for at least certain tumour types. sprou ting blood vessels in the tumour ween ac tive, mature VEGF-C and in - e expression of cathepsin D has long vas culature [58]. ac tive pro-VEGF-C. is can be attri - been correlated with tumour me ta sta - bu ted to the fact that pro-VEGF-C has 3. Tumor cells themselves can ex - sis [64]. Although, in contrast to ca -

94 LymphForsch 23 (2) 2019 REVIEW ARTICLES only been known to be inactive since tives had been achieved [77]. In con - tu mour therapy. In lymphedema and 2014 and that no commercially avai l - trast, the phase 2 studies of Lym fa ctin®, im mune diseases, the typical goal is to able test does distinguish between the sponsored by the Finnish phar ma ceu - increase expression and activation of two forms. For RNA-based expression ti cal start-up Herantis, have just been VEGF-C, whereas in tu mour therapy, analyses (e.g. Gene-Chip®, RNA-Seq) ex pand ed [78]. e two drugs are the goal is to block VEGF-C expression such differentiation is essentially im - based on dif ferent mechanisms of ac - or activation. Balancing these op pos - pos sible, since all VEGF-C forms are tion. Following the observation that ing goals might prove a complex task, trans lated from the same mRNA trans - the anal gesic keto profen relieves par ti cularly in the case of edema which cript of the VEGFC gene. A dif fe ren ti a - lymph edema symp toms in a mouse oc curs as a result of surgical can cer tion of the different VEGF-C forms model [79], the keto profen-like but treat ment. could be achieved with an antibody- more spe cific Besta tin was selected for based test (ELISA, Western blot), but clinical trials [80]. Ketoprofen and such a test has not been developed yet. Bestatin are non-steroidal anti-inflam - More over, the majority of com mer cial - ma tory drugs, and not much detail is Literature ly avai lable antibodies against VEGF-C known about their influence on the 1. Longstaff C, Kolev K: Basic mechanisms and are not even capable of detecting lymphatic sys tem. In contrast, Lym fac - regulation of fibrinolysis. J. Thromb. Haemost. JTH VEGF-C with the necessary sensitivity tin® is a ge ne tically engineered bio - 2015;13 Suppl 1:S98-105. https://onlinelibrary. [42]. It is there fore not surprising that phar ma ceu ti cal that is based on the wiley.com/doi/full/10.1111/jth.12935 the research data are confusing. body’s own VEGF-C production aer 2. Versteeg HH, Heemskerk JWM, Levi M, Reitsma PH: New Fundamentals in Hemostasis. Physiol. Meanwhile, the number of clinical ad mi ni strat ion of a re combinant ade - Rev. 2013;93(1):327–58. https://www.physiology. stu dies that correlate VEGF-C ex pres - no viral vector (AdVEGF-C, see Figure org/doi/full/10.1152/physrev.00016.2011 sion of tumours with the course of the 9), whose me cha nism of action is well 3. Travis MA, Sheppard D: TGF-β Activation and Function in Immunity. Annu. Rev. Immunol. 2014; di sease has exceeded three hundred re searched [81-83]. Depending on the area of ap pli ca tion, the availability of 32(1): 51–82. https://www.annualreviews.org/ (Pub med query: https://mjlab.fi/ doi/10.1146/annurev-immunol-032713-120257 en do ge nous pro te inases and CCBE1 pubmed1). 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Keyt BA, Berleau LT, Nguyen HV, Chen H, Hein - pies ma cological target for several diseases sohn H, Vandlen R, et al.: The Carboxyl-terminal af fecting the im mune system. ese in - Domain(111165) of Vascular Endothelial Growth Although lymphedema can be Factor Is Critical for Its Mitogenic Potency. J. Biol. treated, the aim of research remains a clude chro nic inflammatory bowel di - Chem. 1996;271(13):7788–95. http://www.jbc. org/content/271/13/7788 causal the ra py, because lymph drainage sease [84], psoriasis [85] and rheu ma - 8. Lee S, Jilani SM, Nikolova GV, Carpizo D, Iruela- and ban daging only help to control the to id ar thri tis [17], but also neuro de ge - ne rative di seases such as multiple scle - Arispe ML: Processing of VEGF-A by matrix metal - symp toms of the underlying lymphatic loproteinases regulates bioavailability and vascu - insufficiency. With Bestatin (Ubeni - ro sis and Alz heimer's disease [86]. lar patterning in tumors. J. 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