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Am. J. Trop. Med. Hyg., 77(4), 2007, pp. 601-608
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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Plasma Vascular Endothelial Growth Factor-A (VEGF-A) and VEGF-A Gene Polymorphism are Associated with Hydrocele Development in Lymphatic Filariasis

Alexander Yaw Debrah, Sabine Mand, Mohammad Reza Toliat, Yeboah Marfo-Debrekyei, Linda Batsa, Peter Nürnberg, Bernard Lawson, Ohene Adjei, Achim Hoerauf, AND Kenneth Pfarr*
Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Bonn, Germany; Kumasi Centre for Collaborative Research in Tropical Medicine (KCCR), Kumasi, Ghana; Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana; Cologne Center for Genomics (CCG) and Institute for Genetics, University of Cologne, Germany; School of Medical Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hydrocele is a build-up of fluid in the scrotal regions of a proportion of men infected with the filarial nematode Wuchereria bancrofti. Vascular endothelial growth factors (VEGF) are major mediators of vascular permeability and angiogenesis in the development and progression of many diseases, making them candidates in hydrocele development. We assessed the role of VEGF-A genetic polymorphisms in hydrocele development in a cohort of lymphatic filariasis patients from Ghana. Three VEGF-A promoter polymorphisms were examined. The C/C genotype at –460 was significantly higher in hydrocele patients ([P = 0.0007], OR = 3.8 [95% CI = 1.9–8.2]) than in non-hydrocele patients. Furthermore, plasma levels of VEGF-A were significantly higher in subjects with the C/C genotype than in those with other genotypes. Also, a positive correlation (R2 = 0.412, P = 0.026) was observed between plasma VEGF-A and stage of hydrocele. The data suggest that the C polymorphism at –460 is a genetic risk factor for hydrocele development in lymphatic filariasis.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Filariasis is a major cause of morbidity throughout the tropics. More than 140 million people are infected with the filarial nematodes, Wuchereria bancrofti, Brugia malayi, and Onchocerca volvulus, which are responsible for the majority of human filarial diseases.1,2 Lymphatic filariasis (LF), caused by W. bancrofti, presents a spectrum of clinical states with two major poles.3 One pole is represented by microfilaremic patients with high parasite numbers and down-regulated cell-mediated responses, and the other by patients with lymphedema (LE) and hydrocele, who typically have few or no parasites but vigorous specific immune reactions.3,4 The prevalence of overt clinical manifestations among adult residents of endemic areas is usually less than 10% for lymphedema and 30–50% for hydrocele despite the fact that most individuals are presumably inoculated with infective larvae throughout life.5 In high transmission areas, community-specific infection rates documented by the presence of blood-borne microfilariae (first stage larvae, Mf) and filarial anti-genemia range from 50% to over 80% in persons older than 20 years of age, whereas less than 10% have lymphedema of extremities.5 The actual cause(s) of this heterogeneity in infection and disease is not fully known. However, this has been attributed to differences in inflammatory processes that are immune-mediated,6 secondary bacterial infections superimposed on the lymphatic dysfunction and the immunogenetics of the host.7–9

The completion of the human genome and the sequencing of different populations by HAPMAP have provided invaluable tools to examine the role of allelic variation that are believed to be factors in the development of diseases. Allelic variation has been associated with several infections and autoimmune diseases.10 It is also believed that genetic polymorphism in host immune genes may be major factors in helminth disease development and pathologies. For filariasis, differential susceptibility to infection within a population and within families has been shown.11

The few earlier studies that attempted to address the cause of differential susceptibility to clinical expression of helminth infections implicated mainly major histocompatibility complex (MHC).12–14 However, this may not hold true in all populations.12,13 Cytokine and vascular endothelial growth factor alleles are also known to be involved in manifestations of human infectious and non-infectious diseases such as sepsis, allergy, psoriasis, rheumatoid arthritis, and proliferative retinopathy.15 However, little is known regarding filarial infections. Pathology of filarial infections, which presents as the immunologically hyperreactive state of sowda in onchocerciasis or lymphedema and hydrocele in lymphatic filariasis, is believed to be associated with host immunogenetics.7,9,16

Vascular endothelial growth factors (VEGFs) are a key regulator of endothelial cell functions required for vasculogenesis and also for physiologic and pathologic angiogenesis.17,18 VEGF-A is a major mediator of vascular permeability and angiogenesis, and plays a pivotal role in mediating the development and progression of many diseases such as tumor cancer, diabetic retinopathy, etc.19,20 VEGF-A also promotes extravasation of fluid and plasma proteins, including fibrin, from the blood vessels.21,22 VEGF-A may also be a major angiogenic factor in the development of lymphedema23 and hydrocele in lymphatic filariasis. The binding of VEGF-C and its soluble receptor (sVEGFR-3) is fundamentally important for lymphatic proliferation and it is believed to be involved in lymphatic dilation and lymphedema development in patients with lymphatic filariasis.24 In this study, VEGF-C and sVEGFR-3 were found to be higher in both patients with microfilaremia and lymphedema than endemic normal patients, and sVEGFR-3 was significantly higher in patients with lymphedema than patients with microfilaremia. Preceding clinical improvement, the mean plasma levels of VEGF-C and sVEGFR-3 decreased significantly in patients treated with doxycycline to a level close to that of endemic normal patients, resulting in amelioration of conditions of patients with lymphedema. It was concluded that Wolbachia endobacteria in the filarial worms, which are depleted by doxycycline, may play a major part to the release of VEGFs because the endobacteria are known to induce pro-inflammatory cytokines.6,25

Another report has also shown an association of Chyluria (a filarial disease) with increased levels of serum VEGF-A.23 Because VEGF-A promotes extravasation of fluid and plasma proteins from the blood vessels into the surrounding tissues,21,22 it is conceivable that differential production of VEGF-A by different hosts due to gene variation might play a significant role in the development of hydrocele and lymphedema—conditions characterized by a build-up of fluid in the affected areas. Therefore, we analyzed VEGF-A single nuclear polymorphisms (SNPs) as a possible cause for the different pathologic manifestations in patients with lymphatic filariasis.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study population and classification of groups. The study was carried out in the Nzema East District in the Western region of Ghana, which is an endemic area of lymphatic filariasis. The study was approved by the Ethical Committee on Human Research of the School of Medical Sciences of Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana. The study conformed to the principles of the Helsinki Declaration of 1975 (as revised 1983 and 2004).

In all, three cohorts of 188 unrelated patients with lymphatic filariasis were included in this genotyping analyses. The three groups used in the study are indicated in Table 1Go. Patients with microfilaremia (Mf+) are those with microfilariae in the blood but without any pathology. No other human filarial species were endemic in this area.


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TABLE 1
The age and sex distribution of the patients genotyped
 
All patients included in the study were pricked in the index finger in the night for assessment and quantification of Mf in the blood as described elsewhere.24,26,27 Subsequently, eligible patients donated 10 ml of venous blood for accurate quantification as described previously.24,26 Patients with lymphedema and hydrocele were examined separately by a clinician (S.M.) conversant with the symptoms of lymphatic filariasis using both physical methods as well as ultrasonography (USG) to detect subclinical hydrocele.

Determination of circulating filarial antigenemia. For determination of circulating filarial antigenemia (filarial adult worm antigen, Og4C3), W. bancrofti antigen was measured with the TropBio® ELISA test kit (TropBio, Townsville, Australia). The manufacturer’s protocol was followed; 50 µl of the diluted plasma was added to plate wells in duplicate, and the plates were incubated overnight. The optical density at 414 nm was recorded from the plasma samples. Antigen units were calculated with a standard curve from standards provided by the manufacturer.28

Measurement and staging of hydrocele by ultrasound. Study participants were examined in a supine position using a portable ultrasound machine (SONOSITE® 180 Plus) equipped with a linear transducer (38 mm, 5–10 MHz) as described previously.29 The size (longitudinal and transverse section) of the hydrocele was measured using B-scan ultrasonography measured at a depth of 3.2 cm and converted to stage. Because there was no grading system available for hydrocele, a four-stage grading scheme was developed for this study using ultrasonography as follows:

  1. Stage 1. Sub clinical hydrocele.
  2. Stage 2. The maximal longitudinal and transverse diameters of the hydrocele do not exceed 1.9 and 1.6 cm, respectively (half screen).
  3. Stage 3. The maximal longitudinal and transverse diameters of the hydrocele do not exceed 3.8 and 3.2 cm, respectively (full screen).
  4. Stage 4. The maximal longitudinal and transverse diameters of the hydrocele are greater than 3.8 and 3.2 cm, respectively.

Determination of plasma levels of VEGF-A. The plasma concentration of VEGF-A was measured in patients with microfilaremia, hydrocele, and lymphedema using the Quantikine immunoassay ELISA kit according to the manufacturer’s instructions (R&D Systems, Wiesbaden, Germany). After stopping the reaction, plates were read at 450 nm and 540 nm with a microplate reader (SPECTRAmax® 340PC, Sunnyvale, CA). Plasma levels of VEGF-A of 23 endemic normals (i.e., residents of the same endemic area with no evidence of infection confirmed by the lack of mircofilaremia and lack of circulating Og4C3 filarial antigen despite exposure to infective larvae borne by mosquitoes) were included as controls.

Genotyping. Genomic DNA was extracted from peripheral blood preserved in an equal volume of 8M urea from the patients using the QIAmp® kit from Qiagen (Hilden, Germany) as instructed by the manufacturer. The DNA yielded was measured using an Eppendorf Biophotometer® (Eppendorf AG, Hamburg, Germany) at A260 and A280 nm wavelengths. The A260/A280 ratio was used to determine the purity of the DNA. The DNA concentrations of the samples calculated from the A260 values were corrected to 50 ng/µL.

Three SNPs at –460 C/T (rs833061), –634 C/G (rs2010963), and –1154 A/G (rs1570360) in the promoter region of VEGF-A gene were genotyped. Genotyping of the –460 and –634 polymorphisms was performed using Taqman-based assays that were purchased from Applied Biosystems (Foster City, CA). The PCR was performed with a GeneAmp PCR system, model 9700 (Applied Biosystems). Fluorescence levels of the PCR products were measured with an ABI Prism model 7900 HT sequence detector (Applied Biosytems), which resulted in three clear genotypes of –460 and –634. The third SNP, –1154 A/G, was performed using the pyrosequencing technique as described.30–32 The forward primer was 5'BIOTIN-CTGCATCCTGTCTGGAAGTT-3', the reverse primer was 5'-GCCACTGACTGATTTGTGTG-3', and the internal primer was 5'-GCTGAACCCCGTCC-3'. The PCR amplification used 45 cycles of denaturation at +95°C (30s), annealing at 62°C (30s), and extension at 72°C (30s). The pyrosequencing was performed in a Biotage PSQ HS 96A SNP system (Pyrosequencing AB, Upsala, Sweden), and the data were captured with PSQ HS SNP software as described by Ronaghi.32

Haplotype determination. A haplotype analysis was done and linkage disequilibrium was calculated among the three VEGF-A polymorphisms using SNPAnalyzer v3.1 software.

Statistical analysis. Genotype and haplotype frequencies were summarized as percentages. Frequencies of each polymorphism were tested for Hardy-Weinberg equilibrium. Deviations of the genotype and allele frequencies in the cohorts from those expected under Hardy-Weinberg equilibrium were assessed by Pearson’s goodness fit. Genotype and allele frequencies among the cohorts were compared by {chi}2 test. Linkage disequilibrium and haplotype frequencies for multiple loci were assessed using the SNPAnalyzer v3.1 software available at http://snpwizard.com/snpanalyzer/2.0/. Genotype-specific risks were calculated as odds ratio (OR) and 95% confidence interval (CI) for the OR.

Plasma levels of the VEGF-A were expressed as mean ± standard deviation, and differences in the levels between endemic normals, microfilaremia, and lymphedema patients were analyzed using ANOVA with the Bonferroni/Dunn post hoc test. A positive relationship between the stage of hydrocele and the plasma levels of VEGF-A was assessed by the Spearman’s rank correlation test and simple regression analysis (coefficient of determination indicated as R2). P < 0.05 was considered significant. Independence of data was tested using multivariate analysis. All analyses were done using tests included in the StatView® software version 4.5 for Macintosh.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Clinical characteristics. Of the 188 subjects genotyped, one patient had Mf, hydrocele, and lymphedema at the same time, three patients had both lymphedema and hydrocele, and one patient had both Mf and lymphedema. There was no significant difference in the mean age among the groups (Table 1Go, P = 0.255, ANOVA post hoc, Student’s t test).

Genotype. The frequencies of observed and expected genotypes for each single nucleotide polymorphism and their corresponding P values are shown in the Table 2Go. The observed frequencies of the SNP, VEGF –1154 A/G, were significantly different (P < 0.0001) from the expectations and were not in Hardy-Weinberg equilibrium (HWE), suggesting that there has been some selective pressure on this gene. The other two SNPs (–460 C/T and –634 C/G) were in HWE (Table 2Go).


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TABLE 2
Observed versus expected VEGF-A genotypes for each single nucleotide polymorphism (SNP) in the samples from filarial infected patients
 
Distribution of genotypes in the study populations. Table 3Go shows the genotype frequencies of the three SNPs. Comparing the different subpopulations (Mf+, hydrocele, and lymphedema), no statistically significant differences in the genotypes among the clinical groups were observed for –634 C/G and –1154 A/G (Table 3Go). However, a significant association was found between –460 C/T genotype and hydrocele development (Table 3Go). Carriage of –460 C/T genotype differed significantly between hydrocele and Mf+ patients ([P = 0.0006], OR = 3.8 [95% CI = 1.9–8.0]) and hydrocele and non-hydrocele ([P = 0.0007], OR = 3.8 [95% CI = 1.9–8.2]). However, there was no difference between hydrocele and lymphedema (P = 0.1324). The frequency of –460 C/C homozygous mutant was higher (17%) in hydrocele patients compared with Mf+ patients (5%) and patients with lymphedema (4%) (Table 3Go).


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TABLE 3
Genotype distribution (percent) of VEGF-A gene polymorphisms in lymphatic filariasis-infected patients
 
The –460C allele has been shown to be dominant;20 therefore the analysis was reduced to carriage of the C allele at –460. Reducing the analysis to carriage of the C or T alleles, with the genotype C/T being defined as C, carriage of C was higher in patients with hydrocele (74%) compared with Mf+ (44%) and lymphedema (56%), and there was a significant difference of allele frequencies between hydrocele patients and Mf+ (P = 0.0002). This is an indication that the –460C allele is associated with a higher risk of hydrocele development (OR = 3.8, CI: 1.9–8.2). Thus, patients carrying the allele –460C are on average 3.8 times at higher risk of developing hydrocele than carriers of –460T allele. Because females do not develop hydrocele, the 22 female Mf+ and the 19 female lymphedema patients (Table 1Go) were excluded from the analysis without affecting the significant difference observed between hydrocele and Mf+ (P = 0.0018, OR = 3.6) and hydrocele and lymphedema (P = 0.5430).

Distribution of possible haplotype in the study populations. To assess whether the observed association was attributable to the –460C/T polymorphism alone or together with the two other SNPs in a haplotype, a haplotype analysis was done and possible linkage disequilibrium was calculated among the three VEGF-A polymorphisms using SNP Analyzer.

A significant linkage disequilibrium was found among the three polymorphisms, which confirms other reports,19,33 and the presence of four haplotypes was estimated (Table 4Go). As observed in Table 4Go, the haploytpe –460C/–634C/–1154G (CCG) was significantly higher in hydrocele (P = 0.0084) and lymphedema (P = 0.0253) patients compared with Mf+ patients (P = 0.9876), whereas the haplotype –460T/–634C/–1154G (TCG) was significantly lower in hydrocele patients (0.0283) but not in lymphedema (P = 0.4753) or Mf+ (P = 0.3036) patients. Because these haplotypes differ only in –460C/T position, it can be assumed that the –460 C/T polymorphism is primarily associated with hydrocele among the three polymorphisms and might be the causal variant.


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TABLE 4
Possible Haplotype distribution (percent) of VEGF-A polymorphisms in lymphatic filariasis-infected patients
 
Comparison of plasma levels of VEGF-A in LF-infected patients. The plasma level of VEGF-A was significantly higher in the filarial-infected patients than the endemic normal patients (Figure 1Go), indicating that once a person has LF infection, VEGF-A is expressed at a higher level. More interestingly, the mean plasma levels of VEGF-A was comparatively higher in the hydrocele patients (though not significant) than the rest of the LF-infected groups (Figure 1Go).


Figure 1
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  		FIGURE 1. Plasma levels of VEGF-A in filarial-infected patients and endemic controls. Plasma concentrations (mean ± SD) of VEGF-A were measured, using a commercial kit, from plasma of hydrocele patients (N = 47), lymphedema patients (N = 25), microfilaremic patients (N = 76), and endemic normals (N = 23), who do not have evidence of filarial infection. Mean plasma levels of VEGF-A were significantly elevated in the hydrocele (P = 0.0070), microfilaremic (P = 0.0059), and lymphedema patients (P = 0.0027) compared with endemic normals (Student’s t test with Bonferroni/Dunn correction). There was no difference between hydrocele and microfilaremic (P = 0.62) or hydrocele and lymphedema patients (P = 0.99).

 
Correlation between –460 C/T genotypes and plasma levels of VEGF-A. To assess the possible functional relevance of the –460 C/T polymorphism on the in vivo production of VEGF-A, the plasma levels of VEGF-A and the –460 C/T genotype of the filarial patients (hydrocele, Mf+ and lymphedema) were correlated (Figure 2A–2CGo, respectively). In all the cohorts, the –460 C/C homozygous individuals had the highest plasma VEGF-A levels, followed by C/T heterozygous individuals, whereas the homozygous T/T individuals had the lowest plasma VEGF-A levels (hydrocele P = 0.023; Mf+, P = 0.043; and lymphedema, P = 0.044; Kruskal Wallis test, Figure 2A–2CGo, respectively). A significant difference was also observed between C (C/C and C/T combined) and T/T in all the groups (hydrocele, P = 0.028; Mf+, P = 0.014 and lymphedema, P = 0.038; Student’s t test). The possible functional relevance and the promoter activity of the VEGF-A –460 C/T polymorphism observed in this study, where C/C individuals produce more plasma VEGF-A followed by C/T and least by T/T individuals, conforms to work done by others, which also showed that the promoter carrying the –460C allele has 71% greater activity compared with those that do not have this allele.33


Figure 2
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  		FIGURE 2. A, Association between –460 C/T genotype and plasma levels of VEGF-A in hydrocele patients. Plasma concentrations (mean ± SD) of VEGF-A were measured, using a commercial kit, from plasma of hydrocele patients (N = 47) and correlated with the –460 C/T genotype. Mean plasma levels of VEGF-A were significantly elevated in patients having C/C genotype followed by C/T and least by T/T individuals (P = 0.023, Kruskal Wallis test). A significant difference was also observed between the C allele (C/C and C/T combined) and the T allele (P = 0.028, Student’s t test). B, Association between –460 C/T genotype and plasma levels of VEGF-A in Mf+ patients. Plasma concentrations (mean ± SD) of VEGF-A were measured, using a commercial kit, from plasma of filarial patients (N = 76) and correlated with the –460 C/T genotype. Mean plasma levels of VEGF-A were significantly elevated in patients having C/C genotype followed by C/T and least by T/T individuals (P = 0.043, Kruskal Wallis test). A significant difference was also observed between the C allele (C/C and C/T combined) and the T allele (P = 0.014, Student’s t test). C, Association between –460 C/T genotype and plasma levels of VEGF-A in lymphedema patients. Plasma concentrations (mean ± SD) of VEGF-A were measured using a commercial kit, from plasma of lymphedema patients (N = 25) and correlated with the –460 C/T genotype. Mean plasma levels of VEGF-A were significantly elevated in patients having C/C genotype followed by C/T and least by T/T individuals (P = 0.044, Kruskal Wallis test). A significant difference was also observed between the C allele (C/C and C/T combined) and the T allele (P = 0. 038, Student’s t test).

 
Association of plasma levels of VEGF-A and the stage of hydrocele. To assess the association of plasma levels of VEGF-A with the degree (size) of hydrocele, a correlation test was performed. A positive correlation (R2 = 0.412, P = 0.026) was found between the levels of VEGF-A in the plasma of patients with hydrocele and the stage of hydrocele (Figure 3Go), which confirms an association of VEGF-A and hydrocele development.


Figure 3
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  		FIGURE 3. Plasma levels of VEGF-A positively correlate with stage of hydrocele. A positive correlation (R2 = 0.412, P = 0.026) was observed between the plasma concentrations of VEGF-A in patients with hydrocele and the stage (degree) of hydrocele using Spearman’s rank correlation test and simple regression analysis (coefficient of determination indicated as R2).

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Lymphatic filariasis is ranked as one of the leading causes of permanent disability worldwide by the World Health Organization.34 Infection with W. bancrofti, which leads to development of lymphedema and/or hydrocele—a build-up of fluid due to impaired function of the lymph vessels—occurs in only a proportion of infected persons, even in areas of intense transmission. This amounts to 25 million men suffering from debilitating genital disease (hydrocele), with an additional 15 million people suffering from lymphedema and elephantiasis.34,35 Several observations are consistent with the hypothesis that the proportion of the filarial-exposed population who develop these pathologies may be genetically predisposed.16 Yet, these observations are mainly interpreted from an immunologic perspective because few studies have been done at the molecular level.

In this study, three SNPs in the VEGF-A gene were analyzed in a cohort of lymphatic filariasis-infected patients, and an association was found between the VEGF-A SNP at –460 C/T and hydrocele development, where patients infected with W. bancrofti having the VEGF-A –460 C/C or C/T genotypes have a 3.8 times higher risk of developing hydrocele than those having the T/T genotype. The VEGF-A –634 C/G and VEGF-A –1154 A/G polymorphisms were also analyzed for association with any of the filarial diseases but neither of them was found to be associated with development of filarial pathology.

The data also revealed a positive correlation between the stages of hydrocele and plasma levels of VEGF-A, providing evidence that VEGF-A plays an important role in hydrocele development, and that genetics predisposes an individual to developing hydrocele disease.

Recent data from many publications suggest that VEGF-A has several activities that may amplify acute inflammatory reactions, and it is important in the etiology of several diseases characterized by pathologic angiogenesis such as psoriasis, rheumatoid arthritis, and proliferative retinopathy.15 Deregulated VEGF-A expression also contributes to the development of solid tumors by promoting tumor angiogenesis.36 For instance, over-expression of VEGF-A in the skin results in increased vascular density, enhanced leukocyte adhesion, and tissue infiltration.37

In patients with hydrocele, the actual stimulus responsible for the over-expression of VEGF-A is not yet known. However, VEGF-A is known to be stimulated by inflammatory cytokines such as IL-1B, TNF, IL-4, IL-13, and TGF-ß1.38 These cytokines are in turn stimulated by Wolbachia.6 Studies in animal models for instance, have shown that Wolbachia–derived molecules from Brugia spp. induce pro-inflammatory cytokines including TNF and IL-1B.6 Again, soluble extracts of Brugia and O. volvulus adult and microfilarial worms were also found to stimulate human peripheral mononuclear cells in vitro, resulting in the production of TNF, IL-1B, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-10.25,39 This stimulation was not achieved using extracts from Acanthocheilonema viteae, a filarial species naturally devoid of Wolbachia, and, importantly, with O. volvulus extracts from patients who had been treated with doxycycline to deplete Wolbachia from the worms.40 Thus, it was concluded that in those filarial species that contain these endosymbionts, Wolbachia are the principal stimulating agents for pro-inflammatory cytokines such as TNF, IL-1B, etc.,40 which in turn can stimulate VEGF-A.38 Therefore, it is hypothesized that the Wolbachia in the filarial worm might be responsible for the over-expression of VEGF-A through induction of pro-inflammatory cytokines in the patients with hydrocele.

In our previous studies, targeting the Wolbachia endosymbionts reduced these pro-inflammatory cytokines—namely, TNF, IL-1B, IL-6, etc.—and reduced plasma levels of VEGF-C and soluble VEGF-R3, which resulted in amelioration of lymphatic vessel dilation and lymphedema.24,27 In recent unpublished data, targeting the Wolbachia endosymbionts also led to a macrofilaricidal effect on the filarial worm and reduction of plasma levels of VEGF-A resulting in amelioration of the stage of hydrocele. More interestingly, the amelioration was observed in doxycycline-treated patients with active infection (CFA-positive) but not in the placebo or doxycycline-treated patients without active infection (Debrah, Mand, Hoerauf et al., unpublished data). Therefore, it is hypothesized that the presence or the natural death of the adult worms in the scrotal region of infected people leads to the liberation of filarial and Wolbachia antigens, which in turn leads to over-expression of VEGF-A. The over-expression of VEGF-A molecules in the scrotal region of male patients could be responsible for extravasation and accumulation of fluids, plasma, lymph, etc., from the blood and lymphatic vessels into the scrotal region leading to the formation of hydrocele, chylocele, and lymphocele. It has already been shown that hydrocele development is triggered by the death of the adult worm, which produces an inflammatory nodule that occludes lymphatic vessels,41 and these nodules can persist in a proportion of the infected people. The actual cause of formation of these nodules leading to hydrocele development is not known. The over-expression of VEGF-A, however, could be responsible for the formation of these nodules when DEC is administered.42 This may be similar to the formation of Kaposi’s sarcoma, a multicentric neoplasm consisting of multiple vascular nodules appearing in the skin, mucous membrane, and viscera of AIDS patients,43 which is believed to be caused by over-expression of angiogenic/lymphoangiogenic molecules such as VEGFR-3 and VEGFR-2 (which is also the receptor for VEGF-A).44,45 Lymphatic dysfunction has been shown to be a VEGF-C dependent phenomenon as we have also shown.24 However, pathology of lymphatic filariasis such as lymphedema and hydrocele are multi-factorial, caused by lymphatic dilation and accumulation of fluid in the affected tissues. Therefore, any factor such as VEGF-C/VEGFR-3 (which is responsible for lymphatic dilation) and VEGF-A (which is responsible for extravasation of fluid from the blood and lymphatic vessels into the surrounding tissues) could be involved in a synergistic manner in the manifestation of the disease. VEGF-C is already known to have a synergistic effect with VEGF-A, especially during the induction of angiogenesis. This effect is more prominent in cells expressing both of its receptors.15 Therefore, VEGF-A and VEGF-C could both be involved in the manifestation of pathology of lymphatic filariasis.

On the other hand, there is also hydrocele of non-filarial origin, which can be caused by testicular tumor, scrotal trauma, epididymo-orchitis, etc.46,47 These conditions cause inflammation in the scrotal area leading to the flow of peritoneal fluid into the scrotum. It is not yet known if non-filarial hydrocele is also related to VEGF-A levels. It will therefore be important to investigate this matter.

In conclusion, evidence has been presented that human VEGF-A gene promoter polymorphism is an important risk factor for the development of hydrocele in patients infected with W. bancrofti. This information could be explored in special situations in which either anti-VEGF-A or anti-Wolbachia treatment is administered to patients developing hydrocele to ameliorate or halt this condition in addition to the classic anti-filarial treatment (ivermectin or DEC), which strongly reduces transmission but has no immediate improvement on the pathology in patients, and may even exacerbate the condition by the large release of Wolbachia antigens due to Mf or adult worm killing.42

Received April 30, 2007. Accepted for publication July 5, 2007.

Acknowledgments: We thank the individuals of the District Health Management team at Axim (Nzema East District), Western Region, Ghana for their cooperation.

Financial support: We are grateful for financial support from the European Commission (EU-grant ICA4-CT-2002-10051) and the VW-Foundation (grant 1/81306) to AH, a start-up grant from Bonn Forschung (BONFOR) to KP, and the German Federal Ministry of Sciences and Education (BMBF) through the National Genome Research Network (grant 01GR0416) to PN. AYD is a recipient of a scholarship from the German Academic Exchange Service (DAAD) and of a consumables support grant from BONFOR for his PhD work.

* Address correspondence to Kenneth Pfarr, Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. E-mail: pfarr{at}parasit.meb.uni-bonn.de Back

Authors’ addresses: Alex Y. Debrah, Kumasi Centre for Collaborative Research and Faculty of Allied Health Sciences, Kwame Nkrumah University of Science and Technology, University Post Office, Kumasi, Ghana. Sabine Mand, Achim Hoerauf, and Kenneth Pfarr, Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Sigmund-Freud-Str. 25, Bonn, Germany. Mohamad R. Toliat and Peter Nürnberg, Cologne Centre for Genomics (CCG) and the Institute for Genetics, University of Cologne, Zülpicher-Str. 47, Cologne, Germany. Yeboah Marfo-Debrekyei and Linda Batsa, Kumasi Centre for Collaborative Research, University Post Office, Kumasi, Ghana. Bernard Lawson, Department of Theoretical and Applied Biology, Kwame Nkrumah University of Science and Technology, University Post Office, Kumasi, Ghana. Ohene Adjei, School of Medical Sciences, Kwame Nkrumah University of Science and Technology, University Post Office, Kumasi, Ghana.

Reprint requests: Kenneth Pfarr, Institute for Medical Microbiology, Immunology and Parasitology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Telephone: +49-228-287-11207. Fax: +49-228-287-14330. E-mail: pfarr{at}parasit.meb.uni-bonn.de.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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