Brugia pahangi: Immunization with early L3 ES alters parasite migration,and reduces microfilaremia and lymphatic lesion formation in gerbils (Meriones unguiculatus)

Previous studies have shown that intradermally (ID) injected Brugia pahangi L3s migrate through various tissues and into the lymphatics of gerbils in a distinct pattern. Excretory/secretory products (ES) produced at the time of invasion of B. pahangi are likely to be important in this early migration phase of the parasite life cycle in their rodent host. Hence, early L3 ES was collected from 24 h in vitro cultures of B. pahangi L3 larvae and used in immunization experiments to investigate the effect of immunity to early L3 ES on worm migration, survival and development of B. pahangi. Immunization of gerbils with ES in RIBI adjuvant produced antibodies to numerous ES proteins eliciting a strong humoral response to ES and indirect fluorescent antibody (IFA) assay using anti-ES serum recognized the ES proteins on the surface of B. pahangi L3 larvae. Following ES immunization, gerbils were challenged either ID or intraperitoneally (IP) with 100 L3s of B. pahangi and euthanized at 3 or 106 days post inoculation (DPI). Immunization with early ES slowed the migration of ID inoculated L3 at 3 DPI and significantly altered the locations of adult worms at 106 DPI. Immunization did not induce protection in any treatment group. However, immunized animals had significantly fewer microfilariae per female worm suggesting the antigens in ES are important in microfilariae development or survival in the host. The number of lymphatic granulomas was also significantly reduced in ES immunized animals. It is important to note that microfilariae serve as a nidus in these granulomas. Our results shows immunization with early Brugia malayi L3 ES alters the worm migration, affects circulating microfilarial numbers and reduces lymphatic granulomas associated with B. pahangi infection in gerbils.     

Polyanhydride Nanoparticle Delivery Platform Dramatically Enhances Killing of Filarial Worms

Filarial diseases represent a significant social and economic burden to over 120 million people worldwide and are caused by endoparasites that require the presence of symbiotic bacteria of the genus Wolbachia for fertility and viability of the host parasite. Targeting Wolbachia for elimination is a therapeutic approach that shows promise in the treatment of onchocerciasis and lymphatic filariasis. Here we demonstrate the use of a biodegradable polyanhydride nanoparticle-based platform for the co-delivery of the antibiotic doxycycline with the antiparasitic drug, ivermectin, to reduce microfilarial burden and rapidly kill adult worms. When doxycycline and ivermectin were co-delivered within polyanhydride nanoparticles, effective killing of adult female Brugia malayi filarial worms was achieved with approximately 4,000-fold reduction in the amount of drug used. Additionally the time to death of the macrofilaria was also significantly reduced (five-fold) when the anti-filarial drug cocktail was delivered within polyanhydride nanoparticles. We hypothesize that the mechanism behind this dramatically enhanced killing of the macrofilaria is the ability of the polyanhydride nanoparticles to behave as a Trojan horse and penetrate the cuticle, bypassing excretory pumps of B. malayi, and effectively deliver drug directly to both the worm and Wolbachia at high enough microenvironmental concentrations to cause death. These provocative findings may have significant consequences for the reduction in the amount of drug and the length of treatment required for filarial infections in terms of patient compliance and reduced cost of treatment.     

Immunization of Mastomys coucha with Brugia malayi Recombinant Trehalose-6-Phosphate Phosphatase Results in Significant Protection against Homologous Challenge Infection

Development of a vaccine to prevent or reduce parasite development in lymphatic filariasis would be a complementary approach to existing chemotherapeutic tools. Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine target due to its absence in mammals, prevalence in the major life stages of the parasite and immunoreactivity with human bancroftian antibodies, especially from endemic normal subjects. We have recently reported on the cloning, expression, purification and biochemical characterization of this vital enzyme of B. malayi. In the present study, immunoprophylactic evaluation of Bm-TPP was carried out against B. malayi larval challenge in a susceptible host Mastomys coucha and the protective ability of the recombinant protein was evaluated by observing the adverse effects on microfilarial density and adult worm establishment. Immunization caused 78.4% decrease in microfilaremia and 71.04% reduction in the adult worm establishment along with sterilization of 70.06% of the recovered live females. The recombinant protein elicited a mixed Th1/Th2 type of protective immune response as evidenced by the generation of both pro- and anti-inflammatory cytokines IL-2, IFN-γ, TNF-α, IL-4 and an increased production of antibody isotypes IgG1, IgG2a, IgG2b and IgA. Thus immunization with Bm-TPP conferred considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccine candidate against lymphatic filariasis (LF).     

A Potential Role for the Interaction of Wolbachia Surface Proteins with the Brugia malayi Glycolytic Enzymes and Cytoskeleton in Maintenance of Endosymbiosis

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium of the genus Wolbachia. The Wolbachia represent an attractive target for the control of filarial induced disease as elimination of the bacteria affects molting, reproduction and survival of the worms. The molecular basis for the symbiotic relationship between Wolbachia and their filarial hosts has yet to be elucidated. To identify proteins involved in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other bacterial systems. Two WSP-like proteins (wBm0152 and wBm0432) were localized to various host tissues of the B. malayi female adult worms and are present in the excretory/secretory products of the worms. We provide evidence that both of these proteins bind specifically to B. malayi crude protein extracts and to individual filarial proteins to create functional complexes. The wBm0432 interacts with several key enzymes involved in the host glycolytic pathway, including aldolase and enolase. The wBm0152 interacts with the host cytoskeletal proteins actin and tubulin. We also show these interactions in vitro and have verified that wBm0432 and B. malayi aldolase, as well as wBm0152 and B. malayi actin, co-localize to the vacuole surrounding Wolbachia. We propose that both WSP protein complexes interact with each other via the aldolase-actin link and/or via the possible interaction between the host''s enolase and the cytoskeleton, and play a role in Wolbachia distribution during worm growth and embryogenesis.     

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα^-/-IL-5^-/-) mice. Adult worms generated in IL-4Rα^-/-IL-5^-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα^-/-IL-5^-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα^-/-IL-5^-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development.     

Spontaneous inflammatory pain model from a mouse line with N-ethyl-N-nitrosourea mutagenesis

Background

Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi.

Methods

Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles.

Results and discussion

Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion).

Conclusions

Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.     

Filarial worms reduce Plasmodium infectivity in mosquitoes

Background

Co-occurrence of malaria and filarial worm parasites has been reported, but little is known about the interaction between filarial worm and malaria parasites with the same Anopheles vector. Herein, we present data evaluating the interaction between Wuchereria bancrofti and Anopheles punctulatus in Papua New Guinea (PNG). Our field studies in PNG demonstrated that An. punctulatus utilizes the melanization immune response as a natural mechanism of filarial worm resistance against invading W. bancrofti microfilariae. We then conducted laboratory studies utilizing the mosquitoes Armigeres subalbatus and Aedes aegypti and the parasites Brugia malayi, Brugia pahangi, Dirofilaria immitis, and Plasmodium gallinaceum to evaluate the hypothesis that immune activation and/or development by filarial worms negatively impact Plasmodium development in co-infected mosquitoes. Ar. subalbatus used in this study are natural vectors of P. gallinaceum and B. pahangi and they are naturally refractory to B. malayi (melanization-based refractoriness).

Methodology/Principal Findings

Mosquitoes were dissected and Plasmodium development was analyzed six days after blood feeding on either P. gallinaceum alone or after taking a bloodmeal containing both P. gallinaceum and B. malayi or a bloodmeal containing both P. gallinaceum and B. pahangi. There was a significant reduction in the prevalence and mean intensity of Plasmodium infections in two species of mosquito that had dual infections as compared to those mosquitoes that were infected with Plasmodium alone, and was independent of whether the mosquito had a melanization immune response to the filarial worm or not. However, there was no reduction in Plasmodium development when filarial worms were present in the bloodmeal (D. immitis) but midgut penetration was absent, suggesting that factors associated with penetration of the midgut by filarial worms likely are responsible for the observed reduction in malaria parasite infections.

Conclusions/Significance

These results could have an impact on vector infection and transmission dynamics in areas where Anopheles transmit both parasites, i.e., the elimination of filarial worms in a co-endemic locale could enhance malaria transmission.     

Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi

Background

Lymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7–15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.

Methodology/Principal Findings

We have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.

Conclusions/Significance

We were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident that we can extend our efforts to the construction of strains with further filarial targets (in particular for those species that cannot be cultivated in the laboratory), and perform high-throughput drug screens to identify specific inhibitors of the parasite enzymes. By establishing synergistic collaborations with researchers working directly on different parasitic worms, we aim to aid antihelmintic drug development for both human and veterinary infections.     

Brugia malayi: Effects of nitazoxanide and tizoxanide on adult worms and microfilariae of filarial nematodes

There is an urgent need for safe and effective antifilarials. Prior studies have shown that the nitazoxanide (NTZ) exhibits broad activity against anaerobic bacteria, protozoa, and certain intestinal helminths. We examined the effects of NTZ and tizoxanide (TZ) on Brugia malayi nematodes in vitro and in vivo. In vitro, NTZ and TZ reduced worm motility and viability in a dose-dependent manner. Worm viability was reduced by 50% with both compounds at 2.5 and 20 μg/ml killed adult worms. NTZ or TZ (5 μg/ml) significantly reduced microfilaria release. These compounds blocked worm’s embryogenesis, and decreased microfilarial motility and viability. Treated worms had damaged cuticles and abnormal mitochondria. Wolbachia were not cleared by NTZ or TZ treatment. Neither NTZ nor TZ cleared adult worms or microfilariae in infected gerbils. These results show that NTZ and TZ have potent effects on B. malayi nematodes in vitro. However, they were not effective in vivo.     

Maldevelopment of dermal lymphatics in Wnt5a-knockout-mice

Maintenance of tissue homeostasis and immune surveillance are important functions of the lymphatic vascular system. Lymphatic vessels are lined by lymphatic endothelial cells (LECs). By gene micro-array expression studies we recently compared human lymphangioma-derived LECs with umbilical vein endothelial cells (HUVECs). Here, we followed up on these studies. Besides well-known LEC markers, we observed regulation of molecules involved in immune regulation, acetylcholine degradation and platelet regulation. Moreover we identified differentially expressed WNT pathway components, which play important roles in the morphogenesis of various organs, including the blood vascular system. WNT signaling has not yet been addressed in lymphangiogenesis. We found high expression of FZD3, FZD5 and DKK2 mRNA in HUVECs, and WNT5A in LECs. The latter was verified in normal skin-derived LECs. With immunohistological methods we detected WNT5A in LECs, as well as ROR1, ROR2 and RYK in both LECs and HUVECs. In the human, mutations of WNT5A or its receptor ROR2 cause the Robinow syndrome. These patients show multiple developmental defects including the cardio-vascular system. We studied Wnt5a-knockout (ko) mouse embryos at day 18.5. We show that the number of dermal lymphatic capillaries is significantly lower in Wnt5a-null-mice. However, the mean size of individual lymphatics and the LEC number per vessel are greater. In sum, the total area covered by lymphatics and the total number of LECs are not significantly altered. The reduced number of lymphatic capillaries indicates a sprouting defect rather than a proliferation defect in the dermis of Wnt5a-ko-mice, and identifies Wnt5a as a regulator of lymphangiogenesis.     

Function of Chemokine (CXC Motif) Ligand 12 in Periodontal Ligament Fibroblasts

The periodontal ligament (PDL) is one of the connective tissues located between the tooth and bone. It is characterized by rapid turnover. Periodontal ligament fibroblasts (PDLFs) play major roles in the rapid turnover of the PDL. Microarray analysis of human PDLFs (HPDLFs) and human dermal fibroblasts (HDFs) demonstrated markedly high expression of chemokine (CXC motif) ligand 12 (CXCL12) in the HPDLFs. CXCL12 plays an important role in the migration of mesenchymal stem cells (MSCs). The function of CXCL12 in the periodontal ligament was investigated in HPDLFs. Expression of CXCL12 in HPDLFs and HDFs was examined by RT-PCR, qRT-PCR and ELISA. Chemotactic ability of CXCL12 was evaluated in both PDLFs and HDFs by migration assay of MSCs. CXCL12 was also immunohistochemically examined in the PDL in vivo. Expression of CXCL12 in the HPDLFs was much higher than that in HDFs in vitro. Migration assay demonstrated that the number of migrated MSCs by HPDLFs was significantly higher than that by HDFs. In addition, the migrated MSCs also expressed CXCL12 and several genes that are familiar to fibroblasts. CXCL12 was immunohistochemically localized in the fibroblasts in the PDL of rat molars. The results suggest that PDLFs synthesize and secrete CXCL12 protein and that CXCL12 induces migration of MSCs in the PDL in order to maintain rapid turnover of the PDL.     

In Vitro Silencing of Brugia malayi Trehalose-6-Phosphate Phosphatase Impairs Embryogenesis and In Vivo Development of Infective Larvae in Jirds

Background

The trehalose metabolic enzymes have been considered as potential targets for drug or vaccine in several organisms such as Mycobacterium, plant nematodes, insects and fungi due to crucial role of sugar trehalose in embryogenesis, glucose uptake and protection from stress. Trehalose-6-phosphate phosphatase (TPP) is one of the enzymes of trehalose biosynthesis that has not been reported in mammals. Silencing of tpp gene in Caenorhabditis elegans revealed an indispensable functional role of TPP in nematodes.

Methodology and Principal Findings

In the present study, functional role of B. malayi tpp gene was investigated by siRNA mediated silencing which further validated this enzyme to be a putative antifilarial drug target. The silencing of tpp gene in adult female B. malayi brought about severe phenotypic deformities in the intrauterine stages such as distortion and embryonic development arrest. The motility of the parasites was significantly reduced and the microfilarial production as well as their in vitro release from the female worms was also drastically abridged. A majority of the microfilariae released in to the culture medium were found dead. B. malayi infective larvae which underwent tpp gene silencing showed 84.9% reduced adult worm establishment after inoculation into the peritoneal cavity of naïve jirds.

Conclusions/Significance

The present findings suggest that B. malayi TPP plays an important role in the female worm embryogenesis, infectivity of the larvae and parasite viability. TPP enzyme of B. malayi therefore has the potential to be exploited as an antifilarial drug target.     

A rare case of ocular filariasis caused by Brugia malayi

Extra-lymphatic manifestation of filariasis is uncommon and may usually be clinically misdiagnosed. The extra lymphatic presentation of Brugia malayi may due to the site of entry of the infective larvae, and only a few cases have been proven for the causative species. We report here a 59-year-old woman presented with swollen right conjunctiva and complaint of migratory swelling at her eyelid, which has turned out to be ocular filariasis by B. malayi in Chantaburi province, Thailand. This report highlights the increasing cases of B. malayi as a causative agent of ocular filariasis in human.     

The Orphan Adhesion G Protein-coupled Receptor GPR97 Regulates Migration of Lymphatic Endothelial Cells via the Small GTPases RhoA and Cdc42

The important role of the lymphatic vascular system in pathological conditions such as inflammation and cancer has been increasingly recognized, but its potential as a pharmacological target is poorly exploited. Our study aimed at the identification and molecular characterization of lymphatic-specific G protein-coupled receptors (GPCRs) to assess new targets for pharmacological manipulation of the lymphatic vascular system. We used a TaqMan quantitative RT-PCR-based low density array to determine the GPCR expression profiles of ex vivo isolated intestinal mouse lymphatic (LECs) and blood vascular endothelial cells (BECs). GPR97, an orphan adhesion GPCR of unknown function, was the most highly and specifically expressed GPCR in mouse lymphatic endothelium. Using siRNA silencing, we found that GPR97-deficient primary human LECs displayed increased adhesion and collective cell migration, whereas single cell migration was decreased as compared with nontargeting siRNA-transfected control LECs. Loss of GPR97 shifted the ratio of active Cdc42 and RhoA and initiated cytoskeletal rearrangements, including F-actin redistribution, paxillin and PAK4 phosphorylation, and β1-integrin activation. Our data suggest a possible role of GPR97 in lymphatic remodeling and furthermore provide the first insights into the biological functions of GPR97.     

Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host–parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.     

Phosphorylation of Akt and ERK1/2 is required for VEGF-A/VEGFR2-induced proliferation and migration of lymphatic endothelium

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.