General properties and characteristics

All the cells, tissues and organs of the body are dependent on an efficient supply of O₂ and nutrients, as well as the removal of CO₂ and metabolic waste products (Alitalo et al. Reference Alitalo, Tammela and Petrova2005). These tasks are performed by the circulatory/vascular system. The circulatory system consists of 2 highly branched, tubular networks formed from endothelial cells (ECs), one of which is closed, the primary blood vascular system, and one of which is open, the secondary lymphatic vascular system. Blood circulates through the blood vessels in the direction of arteries-arterioles-capillary beds-venules-veins. The exchange of gases, nutrients and metabolic waste products occurs in the capillaries, and extravasation of macromolecules and cells in the post-capillary venules (Jain, Reference Jain2003; Adams and Alitalo, Reference Adams and Alitalo2007). The lymphatic vascular system functions to maintain fluid balance, in that the blood pressure causes a continuous leakage of plasma into the interstitial space. This extravasated, protein-rich fluid (lymph), along with extravasated leucocytes and activated antigen-presenting cells, is returned via lymphatic capillaries-collecting lymphatic vessels-lymph nodes-lymphatic trunks and ducts to the blood circulation (Alitalo et al. Reference Alitalo, Tammela and Petrova2005).

Development

The development of a functional vascular system is of vital importance in organogenesis as signified by it being one of the earliest events in embryogenesis (Coultas et al. Reference Coultas, Chawengsaksophak and Rossant2005). The initial phase of vasculogenesis is the differentiation and proliferation of ECs in situ and in vivo (i.e., haemangioblasts, haematopoietic and EC precursors; the latter angioblasts migrate and assemble as the outer cells of blood islands). Fusion of these blood islands leads to the appearance of the primary vasculature comprising major vessels, such as dorsal aorta and cardinal veins, and a connecting, primitive capillary plexus. Whereby the capillaries are already specified as, either arterial or venous. The vasculogenic system is characterized as being immature, active and unstable in producing leaky-permeable-haemorrhagic blood vessels (Yancopoulos et al. Reference Yancopoulos, Davis, Gale, Rudge, Wiegand and Holash2000; Karamysheva, Reference Karamysheva2008).

The subsequent phase of angiogenesis, to increase blood flow to the growing tissues, requires the expansion of the pre-existing arteries and veins by circumferential growth and remodelling (arteriogenesis), as well as the sprouting-branching-pruning-remodelling from pre-existing capillaries and post-capillary venules to more complex networks (Carmeliet, Reference Carmeliet2005). Angiogenic sprouting is a complex, morphogenetic event typified by EC sprouting-sprout outgrowth and guidance-sprout fusion and lumen formation-perfusion and maturation (Carmeliet, Reference Carmeliet2003; Adams and Alitalo, Reference Adams and Alitalo2007). The resultant secondary vasculature is a highly organized, hierarchical and stereotyped vascular network, with nascent arteries and veins clad by the differentiated mural cells of vascular smooth muscle cells (VSMCs) and capillaries enveloped by pericytes (Carmeliet, Reference Carmeliet2005; Ferrara and Kerbel, Reference Ferrara and Kerbel2005). This leads to the phenotype of a mature, stable, quiescent and functional blood vascular system.

However, in physiological angiogenesis this adult blood vascular system can still exhibit plasticity, in that, under specific circumstances it can recapitulate developmental angiogenesis’ patterns of growth-branching-pruning and remodelling-repression with ECs displaying cell proliferation-migration-survival-morphogenesis or apoptosis (Jain et al. Reference Jain, Munn and Fukumura2002; Jain, Reference Jain2003). This can occur during wound healing, physiological organ growth and the cyclicity of the female reproductive tract. Pathological angiogenesis, as exemplified by tumours and metastases, shows continual remodelling due to inappropriate patterns of growth and regression to yield an unstable, continually evolving network of abnormal structure and function (Jain, Reference Jain2003).

During embryonic development, lymphangiogenesis occurs by the sprouting of lymphatic endothelial cells (LECs) from embryonic veins, that migrate to lymphatic sacs before growth by sprouting-branching-proliferation-remodelling (Adams and Alitalo, Reference Adams and Alitalo2007). Lymphatic capillaries have a discontinuous basement membrane without mural cells. Collecting lymphatic vessels possess VSMCs, basement membrane and valves (Alitalo et al. Reference Alitalo, Tammela and Petrova2005).

Molecular angiogenic factors

Developmental and physiological angiogeneses are of fundamental importance to the ontogeny and homeostasis of vertebrate physiology. Of necessity, therefore, neovascularization and concomitant, vascular vessel maturation are highly regulated, complex and co-ordinated processes, that require the sequential and balanced activation of a series of specific stimulatory and inhibitory, ligand-receptor signalling pathways (Ferrara et al. Reference Ferrara, Gerber and LeCouter2003). Besides a range of anti-angiogenic factors, the key pro-angiogenic, regulatory system of angiogenesis is the vascular endothelial growth factor (VEGF)-VEGFR (receptor) family-signalling pathway (Ho and Kuo, Reference Ho and Kuo2007; Ferrara, Reference Ferrara2009). However, a number of other ligand-receptor regulators are of major importance: the angiopoietin (Ang)-Tie family; the ephrin B2-EphB4 system; the Delta-like-4 ligand (Dll4)-Notch family signalling cascade; platelet-derived growth factor (PDGFB)-PDGFRβ signal transduction; and transforming growth factor (TGF)β-receptor signalling pathway (Adams and Alitalo, Reference Adams and Alitalo2007; Shibuya, Reference Shibuya2008).

The critical molecule regulating blood vessel-morphogenesis is VEGF-A (synonym, vascular permeability factor [VPF]) and belongs to a family of potent, angiogenic regulators, including, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF). Alternative splicing produces various VEGF-A isoforms, the most important and dominant of which is VEGF-A₁₆₅ (containing 165 amino acids). The heparin binding-induced VEGF-A₁₆₅ concentration gradient promotes EC proliferation-migration-survival-differentiation-sprouting and direct assembly of vascular structures that are necessary for the morphogenetic processes of blood vessel vasculogenesis and angiogenesis (Adams and Alitalo, Reference Adams and Alitalo2007; Karamysheva, Reference Karamysheva2008). This includes the angiogenic capillary sprouting-steps of: vasodilation and plasma protein-extravasation formation of a provisional matrix, containing fibronectin and fibrin etc., for activated EC migration; extra-cellular matrix (ECM) remodelling through EC-mediated, pericellular proteolysis by surface-bound and secreted proteases of the matrix metalloproteinase (MMP2, MMP9 and MT1-MMP) and plasminogen activator-plasmin systems (Pepper, Reference Pepper2001; van Hinsbergh and Koolwijk, Reference Van Hinsbergh and Koolwijk2008); differential expression of tip-cell sprouts; and induction of the angiogenic switch i.e. of EC invasiveness and motility (Bergers and Benjamin, Reference Bergers and Benjamin2003). Signalling is mainly through the EC-located receptor tyrosine kinase, VEGFR2. Under specific circumstances, such as embryonic/developmental angiogenesis, this signal is negatively modulated by VEGFR1 binding of the EC-located receptor. PlGF, a specified VEGFR1-specific ligand, only shows an enhancement of neovascularization during pathological angiogenesis i.e. tumour growth (Carmeliet, Reference Carmeliet2003) and primary tumour metastasis (Hiratsuka et al. Reference Hiratsuka, Nakamura, Iwai, Murakami, Itoh, Kijima, Shipley, Senior and Shibuya2002; Shibuya, Reference Shibuya2006). The key molecule regulating lymphatic vessel morphogenesis (lymphangiogenesis) is VEGF-C, signalling through the EC-located VEGFR3 (Adams and Alitalo, Reference Adams and Alitalo2007). Signal amplification is dependent on co-receptor neuropilins (NRP), which are differentially distributed on arteries (NRP1), and veins and lymphatics (NRP2). The upregulation of many angiogenic genes, primarily the induction of VEGF-A, is triggered and driven by hypoxia. The tissue- and organ growth-generated, hypoxic conditions are signalled through hypoxia-inducible transcription factors (Ferrara et al. Reference Ferrara, Gerber and LeCouter2003).

The vascular endothelium-located, Dll4 (transmembrane ligand)-Notch receptor signalling system is responsible for the complex cascade involved in selection of the non-proliferating tip cell-phenotype from a subpopulation of ECs engaged in angiogenic capillary sprouting. The Notch pathway is also required for formation of the arterial branch during arteriovenous differentiation (Adams and Alitalo, Reference Adams and Alitalo2007; Karamysheva, Reference Karamysheva2008).

The ephrin B2 transmembrane ligand serves as an arterial EC marker, whilst the cognate tyrosine kinase receptor EphB4 is a venous EC marker. The ephrin B2-EphB4 pathway is essential for angiogenesis, in that it is also concerned with the establishment of arterial and venous identity (Carmeliet, Reference Carmeliet2003; Adams and Alitalo, Reference Adams and Alitalo2007). However, the ultimate regulator of these arterial differentiation pathways is VEGF-A. In addition, EC specialization can be organ-specific with the finding that endocrine gland EC-capillary, pore-like fenestrations are induced by the tissue-specific, angiogenic regulator, endocrine gland (EG)-VEGF (LeCouter et al. Reference LeCouter, Kowalski, Foster, Hass, Zhang, Dillard-Telm, Frantz, Rangell, DeGuzman, Keller, Peale, Gurney, Hillan and Ferrara2001).

These nascent, immature and unstable blood vessels are stabilized by mural cell recruitment and ECM formation (Jain, Reference Jain2003). This process is organized by the Ang1-Tie2, PDGFB-PDGFRβ and TGFβ signalling systems. PDFGB plays the key role in pericyte recruitment. The EC-secreted PDFGB facilitates both mural cell proliferation, differentiation and migration-recruitment via interaction with the latter cell type's PDGFRβ-receptor tyrosine kinase (Karamysheva, Reference Karamysheva2008). Pericyte- and VSMC-secreted Ang1 interacts with the EC-located receptor tyrosine kinase-Tie2, to promote pericyte-endothelium association and to lower vascular permeability (Karamysheva, Reference Karamysheva2008). TGFβ1, a pericyte- and EC-derived, multifunctional cytokine, contributes to blood vessel maturation with ECM production and mural cell differentiation (Jain, Reference Jain2003). Less is known on lymphatic vessel maturation, but it is acknowledged to involve the angiopoietins and ephrin B2 (Alitalo et al. Reference Alitalo, Tammela and Petrova2005).

The autocrine, EC-expressed Ang2-Tie2 system has different roles in developmental as opposed to physiological and pathological angiogeneses. In embryonic angiogenesis, Ang2 is a competitive inhibitor of Ang1, i.e. a Tie2 antagonist, to block its vascular, stabilizing action. In physiological and pathological angiogeneses, Ang2 has a key role in destabilizing the vasculature as a necessary prerequisite to subsequent remodelling. The destabilized blood vessels have 2 fates depending on the presence or absence of VEGF. If VEGF is absent vascular regression and EC apoptosis ensue, whilst in the presence of VEGF Ang2 supports renewed angiogenesis (Yancopoulos et al. Reference Yancopoulos, Davis, Gale, Rudge, Wiegand and Holash2000; Karamysheva, Reference Karamysheva2008).

A number of endogenous, anti-angiogenic factors have been reported, for example, thrombospondin-1 (TSP-1; Rodriguez-Manzaneque et al. Reference Rodriguez-Manzaneque, Lane, Ortega, Hynes, Lawler and Iruela-Arispe2001), angiostatin (O'Reilly et al. Reference O'Reilly, Holmgren, Shing, Chen, Rosenthal, Moses, Lane, Cao, Sage and Folkman1994, Reference O'Reilly, Holmgren, Chen and Folkman1996) and endostatin (O'Reilly et al. Reference O'Reilly, Boehm, Shing, Fukai, Vasios, Lane, Flynn, Birkhead, Olsen and Folkman1997). TSP-1 is anti-angiogenic, in that it inhibits EC proliferation and migration, and induces EC apoptosis. Angiostatin and endostatin belong to the ‘statins’, inhibitory molecules derived by specific cleavage from larger proteins, which themselves are inactive. Thus, angiostatin is a plasminogen fragment and endostatin is a C-terminal fragment of collagen type XVIII.

Tumours are also dependent on a blood supply thereby neovascularization is usually considered a prerequisite for tumour growth, progression and metastasis. It is, therefore, imperative for the tumour to gain access to the host's vascular system and generate a tumour vasculature. The induction of the tumour vasculature is termed the angiogenic switch (Bergers and Benjamin, Reference Bergers and Benjamin2003) and is characterized, among other things, by an imbalance of pro- and anti-angiogenic factors. The loss of tight regulation results in tumour vessels that are unstable (due to altered EC-pericyte interactions)-immature-non-quiescent-haemorrhagic-leaky, in part, due to VEGF-A overproduction (Carmeliet, Reference Carmeliet2000, Reference Carmeliet2003, Reference Carmeliet2005). In the following review, we wish to develop and reinforce our working hypothesis as being analogous to that of tumour angiogenesis; that successful, metazoan parasitism of growth, maturation, egg production and reinfection are reliant on parasitic helminth-associated angiogenesis, which expresses, at the least, some of the features of neovascularization-dependent tumorigenesis.

At least 2 pro-angiogenic components of the angiogenic switch have been found to be VEGF-A (Inoue et al. Reference Inoue, Hager, Ferrara, Gerber and Hanahan2002) and MMP9 (Bergers et al. Reference Bergers, Brekken, McMahon, Vu, Itoh, Tamaki, Tanzawa, Thorpe, Itohara, Werb and Hanahan2000; Hiratsuka et al. Reference Hiratsuka, Nakamura, Iwai, Murakami, Itoh, Kijima, Shipley, Senior and Shibuya2002). Induced and upregulated MMP9 is a functional constituent of the angiogenic switch in both carcinogenesis and primary tumour metastasis, either by increasing the bioavailability of ECM-sequestered VEGF or preparing distant, premetastatic niches for specific, neoplastic cell invasion, respectively. An anti-angiogenic component of the angiogenic switch in tumour progression has been found to be the endogenous angiogenesis inhibitor TSP-1 (Rodriguez-Manzaneque et al. Reference Rodriguez-Manzaneque, Lane, Ortega, Hynes, Lawler and Iruela-Arispe2001). TSP-1 upregulation was correlated with the suppression of spontaneous tumour growth and the inhibition of MMP9 activation, with the consequent prevention of VEGF mobilization from ECM sequestration, for example, perlecan (Jiang and Couchman, Reference Jiang and Couchman2003) for VEGFR2-mediated transduction (Fig. 1).

Fig. 1. Hypothesized pathway(s) of parasitic helminth-associated (lymph)angiogenesis. (A) Partition of (lymph)angiogenesis into four developmental stages: I, release of helminth- and/or upregulation of host-derived, proangiogenic factors; II, angiogenic switch of endothelial cell activation; III, remodelling events of parasite vasculature-formation; IV, maintenance of immaturity or maturation of parasite vasculature. (B) Cartoon of the four phases of (lymph)angiogenesis: I, priming of vascular or lymphatic, endothelial cells by parasitic helminth- and/or inflammatory infiltrate-generated, proangiogenic factors; II, angiogenic switch and focal breakdown of vessel-basement membrane; III, remodelling and formation of parasite-determined vasculature; IV, maintenance of immaturity or pericyte-identified maturation. ECs, vascular endothelial cells; ECM, extracellular matrix; FGF, fibroblast growth factor; LECs, lymphatic endothelial cells; MMPs, matrix metalloproteinases; VEGF, vascular endothelial growth factor.

Cestoda

The only previous systematic studies of the Cestoda have been carried out on the metacestode larval stage in intermediate hosts of Mesocestoides corti, present in mice and the ‘pork’ tapeworm, Taenia solium, in humans and pigs. This cyst stage can develop in the central nervous system (CNS) as a causative agent of neurocysticercosis. The resultant, protective, granulomatous immune response surrounding the lesion(s) is characterized by an inflammatory blood immune cell-infiltrate, fibrosis, a loss of blood-brain barrier (BBB) function, i.e. permeability, and wound healing (Restrepo et al. Reference Restrepo, Alvarez, Castano, Arias, Restrepo, Trujillo, Colegial and Teale2001). The wound-healing response involved astrocyte-dependent, glial scar formation, fibroblast-derived collagen type I deposition and angiogenesis (Alvarez et al. Reference Alvarez, Colegial, Castano, Trujillo, Teale and Restrepo2002). The rapid neovascularization of astrocyte endfeet-deficient blood vessels and enhanced BBB permeability is associated with the upregulation of astrocytes’, parenchymal vessels’ and inflammatory blood immune cells’ VEGF expression (Alvarez and Teale, Reference Alvarez and Teale2006; Sikasunge et al. Reference Sikasunge, Johansen, Phiri, Willingham and Leifsson2009). This was accompanied by the neovascular and perivascular localization of mast cell-juxtaposed, basic fibroblast growth factor (FGF2; Alvarez et al. Reference Alvarez, Colegial, Castano, Trujillo, Teale and Restrepo2002), a known vasoactive and pro-fibrotic cytokine. The FGF family of heparin-binding, pleiotropic growth factors, among other properties, participate in both adult physiological and pathological angiogeneses, through cross-talk and synergism with VEGF-VEGFR system activation (Presta et al. Reference Presta, Dell'Era, Mitola, Moroni, Ronca and Rusnati2005). Even cases of intraocular, neovascular glaucoma (=neovascularization), with surgical and anti-VEGF MAb treatments, have been demonstrated to be due to T. solium metacestode cysts (Ratra et al. Reference Ratra, Phogat, Singh and Choudhari2010).

These findings have been interpreted as supporting the proposed, working hypothesis of a parasitic helminth-provoked, host-elicited angiogenic response. The larval stage of the fox tapeworm, Echinococcus multilocularis (human alveolar echinococcosis), in the intermediate host's liver, exhibits a tumour-like, infiltrative growth of the multivesicular metacestodes through asexual reproduction and is surrounded by newly formed blood vessels in the granulation tissue (Guerret et al. Reference Guerret, Vuitton, Liance, Pater and Carbillet1998; Weiss et al. Reference Weiss, Bauer and Kohler2010). From a molecular and functional comparison with the multifunctional, mammalian protein phosphoglucose isomerase (PGI), both parasitic growth and parasite-associated angiogenesis involve the highly conserved E. multilocularis analogue (EmPGI; (Stadelmann et al. Reference Stadelmann, Spiliotis, Muller, Scholl, Muller, Gottstein and Hemphill2010). It can be speculated that the pro-angiogenic potential of EmPGI is not only required for neovascularization, but is responsible, at least in part, for the tumorigenic- and metastatic-like growth of E. multilocularis metacestodes.

Nematoda

Trematoda

Most investigations of parasitic helminth-associated neovascularization have been carried out on the definitive hosts of intravascular members of the Trematoda. For the adult Unitubulotestis sardae in the teleost Sarda sarda, an inflammatory infiltrate was accompanied by a gill arch-originating network of anastomosing, variably sized blood vessels (Marino et al. Reference Marino, Giannetto, Cavallaro, Paradiso, Bottari and De Vico2003). Avian schistosomiasis in the swan Cygnus cygnus, a probable infection by Trichobilharzia spp., revealed vascular lesions of venous hypertrophy distinguished by an intense medial VSMC proliferation with eventual occlusion (Akagami et al. Reference Akagami, Nakamura, Nishino, Seki, Shimizu and Yamamoto2010).

Cat and Syrian hamster models of the American lung fluke, Paragonimus kellicotti (American paragonimiasis), displayed an early, pulmonary pathogenesis of intense, inflammatory granulomas and fibrosis, which was accompanied by significant alterations in vascularity. This remodelling comprised pleural neovascularization, tunica media hyperplasia and hypertrophy of pulmonary arterioles, arteries and veins, and increased vascular permeability (Weina and England, Reference Weina and England1990; Weina and Burns, Reference Weina and Burns1992). Histopathology of late, not early, bovine fascioliasis due to Fasciola hepatica liver infection is characterized by vascular modification in portal and hepatic veins and, in particular, hepatic arteries. However, due to the temporal separation from infection does this transformation represent indirect and secondary, rather than direct and primary, effects of parasitism (Shirai et al. Reference Shirai, Sato, Shibuya, Naito and Tsukise2006)?

Because of their pathobiology, we have decided to include in this review the oriental liver fluke, Opisthorchis viverrini (opisthorchiasis), as well as the closely related Chinese liver fluke, Clonorchis sinensis (clonorchiasis). The diseases are distinguished by chronic inflammation, cholangiocyte proliferation and fibrosis of the target organ, intra- and extrahepatic bile ducts. Opisthorchiasis and, to a lesser extent, clonorchiasis are associated with cholangiocarcinogenesis e.g. cholangiocarcinoma, a cancer of the bile duct (Sripa et al., Reference Sripa, Kaewkes, Sithithaworn, Mairiang, Laha, Smout, Pairojkul, Bhudhisawasdi, Tesana, Thinkamrop, Bethony, Loukas and Brindley2007, Reference Sripa, Bethony, Sithithaworn, Kaewkes, Mairiang, Loukas, Mulvenna, Laha, Hotez and Brindley2010). They are representatives of the so-called group of carcinogenic, metazoan helminths (Mayer and Fried, Reference Mayer and Fried2007). For O. viverrini, a putative, secreted, pathogenic growth factor has been identified by proteomic analysis as an orthologue of human granulin (Smout et al. Reference Smout, Laha, Mulvenna, Sripa, Suttiprapa, Jones, Brindley and Loukas2009), and one of the properties of granulin believed to promote carcinoma progression is the angiogenesis potential! We consider these data, along with investigation of this liver fluke's transcriptome (Laha et al. Reference Laha, Pinlaor, Mulvenna, Sripa, Sripa, Smout, Gasser, Brindley and Loukas2007; Young et al. Reference Young, Campbell, Hall, Jex, Cantacessi, Laha, Sohn, Sripa, Loukas, Brindley and Gasser2010), warrant the inclusion of O. viverrini among the ‘hot-spot species’ group with significance and relevance to the topic parasitic helminth-associated neovascularization.

Urinary schistosomiasis (Schistosoma haematobium) is a major disease of humans, because infection is strongly correlated with the prevalence of bladder-adenocarcinoma malignancy. Along with lymph node metastasis, tumour angiogenesis as quantified by microvessel density is being used as a prognostic marker of carcinogenesis (El-Sobky et al. Reference El-Sobky, Gomha, El-Baz, Abol-Enein and Shaaban2002; Elsobky et al. Reference Elsobky, El-Baz, Gomha, Abol-Enein and Shaaban2002). The disease itself is delimited by inflammatory infiltrates, granulomas surrounding eggs and periovular angiogenesis within the portal tract of the liver, as well as the urinary bladder (Botros et al. Reference Botros, Hammam, El-Lakkany, El-Din and Ebeid2008). The EC mitogenic activity of the egg-generated, soluble schistosome egg antigen (SEA) has been proposed to be due to the presence of proangiogenic growth factors (El-Awady et al. Reference El-Awady, Gad, Wen, Eassawi, Effat, Amr, Ismail and Christ2001).

Most of the work on SEA-induced neovascularization, however, has been performed on intestinal schistosomiasis (Schistosoma mansoni). The characteristic trait of both S. mansoni and S. haematobium infections is the periovular granuloma in the intestine, liver and/or bladder. The resultant, inflammatory response culminates in the activation of blood leucocytes, fibroblasts and ECs, whereby the wound-healing reaction is co-ordinated with an earlier angiogenesis and a later fibrogenesis (Lenzi et al. Reference Lenzi, Sobral and Lenzi1988; Baptista and Andrade, Reference Baptista and Andrade2005; Van de Vijver et al. Reference Van de Vijver, Colpaert, Jacobs, Kuypers, Hokke, Deelder and Van Marck2006), or concomitant geneses as in the rodent, Calomys callosus (Lenzi et al. Reference Lenzi, Mota, Pelajo-Machado, Vale, Vale, Andrade and Lenzi2002). This is a probable example of EC-inflammatory immune cell cross-talk evident in the physiology and pathophysiology of inflammation-associated angiogenesis i.e., of angiogenesis-inflammation and angiogenesis-tumour-inflammation cross-talk (Mor et al. Reference Mor, Quintana and Cohen2004; Noonan et al. Reference Noonan, De Lerma Barbaro, Vannini, Mortara and Albini2008). The nascent blood vessels bridging the periovular granuloma-occluded, pre-sinusoidal capillaries in the interlobular spaces of the liver are mainly of arterial, as well as of venous origin, but are aberrant in being tortuous, thin-walled, dilated, congested and varicose (Bogliolo, Reference Bogliolo1957; Andrade and Cheever, Reference Andrade and Cheever1971; Bloch et al. Reference Bloch, Wahab and Warren1972). Pathogenesis, pathophysiology and vascular remodelling are all more severe in hepatic lesions of periportal-pipestem fibrosis in hepatosplenic, as opposed to hepato-intestinal schistosomiasis (Silva et al. Reference Silva, Ribeiro-Dos-Santos, Soares and Andrade2006).

Earlier findings had shown ECs to be responsive to, in particular, pro-inflammatory cytokine-and parasite contact-mediated activation, involving the generation of and NO-dependent killing of schistosomula (Oswald et al. Reference Oswald, Eltoum, Wynn, Schwartz, Caspar, Paulin, Sher and James1994a,Reference Oswald, Wynn, Sher and Jamesb), migration and attachment during the active extravasation of eggs (Ngaiza et al. Reference Ngaiza, Doenhoff and Jaffe1993; File, Reference File1995), upregulation of the cell-adhesion molecules, intercellular adhesion molecule (ICAM)-1, E-selectin and vascular cell adhesion molecule (VCAM)-1 during egg attachment (Ritter and McKerrow, Reference Ritter and McKerrow1996; Lejoly-Boisseau et al. Reference Lejoly-Boisseau, Appriou, Seigneur, Pruvost, Tribouley-Duret and Tribouley1999), and presentation of an anti-inflammatory phenotype in cerebral capillary ECs by lung-stage schistosomula (Trottein et al. Reference Trottein, Descamps, Nutten, Dehouck, Angeli, Capron, Cecchelli and Capron1999). In addition, the angiogenic phenotype during periovular granuloma morphogenesis requires the full-spectrum of activated EC activities, including, proliferation (Andrade, Reference Andrade2009), upregulation of cell-adhesion molecules (Esterre et al. Reference Esterre, Plichart, Huin-Blondey and Nguyen2005) and previous, focal remodelling of the ECM (Singh et al. Reference Singh, Gerard, Hudson and Boros2004).

Specifically, egg granuloma-derived and -secreted factors were found to stimulate fibroblast and VSMC proliferation (Dunn et al. Reference Dunn, Mansour, Guindy and Rojkind1986; Wyler et al. Reference Wyler, Prakash and Libby1987), as well as material that promoted EC growth (Wyler et al. Reference Wyler, Prakash and Libby1987). These inflammatory immune cell-elicited activities were assumed to be involved in the pathogenesis of hepatic fibrosis and compensatory neovascularization of fibrous scar tissue, respectively. Thus, fibrosin is the CD4+ T-lymphocyte- and fibroblast-generated, novel, fibrogenic cytokine modulating tissue fibrosis as a complication of schistosomiasis mansoni-induced, chronic inflammation (Wyler, Reference Wyler1996; Prakash et al. Reference Prakash, Paul and Robbins2007). In the absence of inflammatory immune cells, SEA still mediated direct effects on fibroblast (Wyler and Tracy, Reference Wyler and Tracy1982) and EC (Freedman and Ottesen, Reference Freedman and Ottesen1988) proliferation. Such EC-stimulating activity in vitro (mitogenic) frequently translates into a pro-angiogenic growth factor(s) in vivo. The SEA-mediated activator of vascular ECs supported this conclusion in being anti-apoptotic, and capable of both proliferation and in vitro angiogenesis i.e., differentiation as defined by capillary-tube formation (Loeffler et al. Reference Loeffler, Lundy, Singh, Gerard, Hudson and Boros2002). The angiogenic switch was elicited by upregulation of EC VEGF and FGFR2, but not of FGF2, FGFR1, VEGFR1 or VEGFR2. Our own work has specified a second, direct-acting, egg-derived, pro-angiogenic factor, whose biological properties were concluded as not being homologous to VEGF and whose biochemical properties were characteristic, in part, of a highly glycosylated moiety (Kanse et al. Reference Kanse, Liang, Schubert, Haas, Preissner, Doenhoff and Dennis2005; Dennis et al. Reference Dennis, Geyer, Geyer and Kamerling2007). Whatever the geneses of the egg-induced, pro-angiogenic factor-mediated neovascularization of periportal fibrotic tissue, the angiogenic phenotype encompasses MMPs and TIMPs, in particular MMP9, in the pathophysiology of acute granulomatous and fibrotic responses at 9 weeks post-infection (Vaillant et al. Reference Vaillant, Chiaramonte, Cheever, Soloway and Wynn2001; Singh et al. Reference Singh, Gerard, Hudson and Boros2004). As a biomedical application, circulating levels of VEGF and anti-SEA IgG4 have been assessed as markers of disease progression and development of periportal fibrosis, respectively (Tawfeek et al. Reference Tawfeek, Alafifi and Azmy2003).

As a prelude to N-terminal tag sequence-characterization, as well as the standardized protocols of tryptic in-gel digestion, peptide mass fingerprinting, mass spectrometric fragmentation analysis and database searches (Galuska et al. Reference Galuska, Rollenhagen, Kaup, Eggers, Oltmann-Norden, Schiff, Hartmann, Weinhold, Hildebrandt, Geyer, Muhlenhoff and Geyer2010) etc., the isolation of the pro-angiogenic factor has proved particularly intractable. In this case, firstly, samples have been re-tested with a second in vitro angiogenesis system, the EC spheroid assay (Fig. 2; Korff and Augustin, Reference Korff and Augustin1998, Reference Korff and Augustin1999). Secondly, repetitive fractionation by multiple runs on C₁₈ reverse-phase HPLC and screening with VSMCs demonstrated the loss of biological activity. Preliminary data with a re-pooled, fractionated aliquot have been interpreted as being a result of synergism (unpublished results), whereby biological activity consists of more than one component, for example, the male mating and dauer pheromones of C. elegans (Edison, Reference Edison2009). Thirdly, the future use of polymyxin B-bound beads to avoid any LPS contamination and the liability of upregulated VEGF expression (Marx et al. Reference Marx, Mulder, Van Den Bergh, Overbeeke, Haanen and Vermes1999).

Fig. 2. Response of endothelial cells to SEA as verified by the spheroid assay. (A) Response of BRECs to high levels of SEA (10 μg/ml): 1, negative control; 2, positive control FGF2 at 5 ng/ml; 3, intestinal SEA; 4, hepatic SEA; 5, adult worm extract. Calibration bar=500 μm. (B) Response of HUVECs to high levels of SEA (10 μg/ml): 1, negative control; 2, positive control FGF2 at 5 ng/ml; 3, intestinal SEA; 4, hepatic SEA; 5, adult worm extract. Calibration bar=500 μm. (C) Analysis in vitro of capillary total-length formation by spheroid assay at high and low levels of SEA (10 and 0·1 μg/ml). A II, adult worm extract of batch II; BRECs, bovine retinal endothelial cells; FGF, fibroblast growth factor; HUVECs, human umbilical vein endothelial cells; SEA, soluble schistosome egg antigen; SI II/IV, intestinal SEA of batches II/IV; SL III/IV, hepatic SEA of batches III/IV; VEGF, vascular endothelial growth factor as positive control at 100 ng/ml.