Doxycycline reduces plasma VEGF-C/sVEGFR-3 and improves pathology in lymphatic filariasis

Abstract

Lymphatic filariasis is a disease of considerable socioeconomic burden in the tropics. Presently used antifilarial drugs are able to strongly reduce transmission and will thus ultimately lower the burden of morbidity associated with the infection, however, a chemotherapeutic principle that directly induces a halt or improvement in the progression of the morbidity in already infected individuals would constitute a major lead. In search of such a more-effective drug to complement the existing ones, in an area endemic for bancroftian filariasis in Ghana, 33 microfilaremic and 18 lymphedema patients took part in a double-blind, placebo-controlled trial of a 6-wk regimen of 200 mg/day doxycycline. Four months after doxycycline treatment, all patients received 150-200 microg/kg ivermectin and 400 mg albendazole. Patients were monitored for Wolbachia and microfilaria loads, antigenemia, filarial dance sign (FDS), dilation of supratesticular lymphatic vessels, and plasma levels of lymphangiogenic factors (vascular endothelial growth factor-C [VEGF-C] and soluble vascular endothelial growth factor receptor-3 [(s)VEGFR-3]). Lymphedema patients were additionally monitored for stage (grade) of lymphedema and the circumferences of affected legs. Wolbachia load, microfilaremia, antigenemia, and frequency of FDS were significantly reduced in microfilaremic patients up to 24 mo in the doxycycline group compared to the placebo group. The mean dilation of supratesticular lymphatic vessels in doxycycline-treated patients was reduced significantly at 24 mo, whereas there was no improvement in the placebo group. Preceding clinical improvement, at 12 mo, the mean plasma levels of VEGF-C and sVEGFR-3 decreased significantly in the doxycycline-treated patients to a level close to that of endemic normal values, whereas there was no significant reduction in the placebo patients. The extent of disease in lymphedema patients significantly improved following doxycycline, with the mean stage of lymphedema in the doxycycline-treated patients being significantly lower compared to placebo patients 12 mo after treatment. The reduction in the stages manifested as better skin texture, a reduction of deep folds, and fewer deep skin folds. In conclusion, a 6-wk regimen of antifilarial treatment with doxycycline against W. bancrofti showed a strong macrofilaricidal activity and reduction in plasma levels of VEGF-C/sVEGFR-3, the latter being associated with amelioration of supratesticular dilated lymphatic vessels and with an improvement of pathology in lymphatic filariasis patients.

Figure 1. Flowchart of Patient Participation

(A) Trial profile of microfilaremic patients. Of the 76 patients, 33 (17 doxycycline and 16 placebo) patients were present at all time points. (B) Trial profile of lymphedema patients. Of the 19 patients, 18 (eight doxycycline and ten placebo) patients were present at all time points.

Figure 2. The Effect of Doxycycline Treatment on the Supratesticular Lymphatic Vessel Dilation at Various Time Points

The supratesticular lymphatic vessel dilation (mean of category ± SD) was determined before treatment, and 12 and 24 mo thereafter, using USG. The mean supratesticular lymphatic vessel dilation of doxycycline-treated (Doxy) patients improved significantly compared to pretreatment (n = 9, p = 0.0404) at 24 mo, in contrast to the placebo group (n = 8) (paired t-test).

Figure 3. Pretreatment Plasma Levels of VEGF-C in Filarial-Infected Patients and Endemic Controls

Plasma concentrations (mean ± SD) of VEGF-C were measured, using a commercial kit, from plasma of lymphedema patients (n = 26), microfilaremic patients (n = 76), and endemic controls (n = 23, who did not have filarial infection). Mean plasma levels of VEGF-C were significantly elevated in the microfilaremic (p < 0.0001) and lymphedema patients (p = 0.0002) compared to endemic controls (Student t-test with Bonferroni/Dunn correction). There was no difference between microfilaremic and lymphedema patients (p = 0.8033).

Figure 4. Pretreatment Plasma Levels of sVEGFR-3 in Filaria-Infected Patients and Endemic Controls

Plasma concentrations (mean ± SD) of sVEGFR-3 were measured using a commercial kit from plasma of lymphedema patients (n = 26), microfilaremic patients (n = 76), and endemic controls (n = 23, who did not have filarial infection). Mean plasma levels of sVEGFR-3 were significantly elevated in the microfilaremic (p = 0.0006) and lymphedema patients (p = 0.0012) compared to endemic controls (Student t-test with Bonferroni/Dunn correction). sVEGFR-3 was also significantly elevated in lymphedema patients (p = 0.0024) compared to microfilaremic patients.

Figure 5. Plasma Levels of VEGF-C of Microfilaremic Patients before and 12 Mo after Doxycycline Treatment

Plasma concentrations (mean ± SD) of VEGF-C were measured from plasma of microfilaremic patients before and 12 mo after doxycycline treatment (17 doxycycline treated, 16 placebo, see Table 1). The VEGF-C levels decreased significantly at 12 mo (preceding supratesticular lymphatic dilation, see Table 2) in the doxycycline-treated patients (p = 0.0198), but no difference in the placebo group occurred (paired t-test).

Figure 6. Plasma Levels of sVEGFR-3 of Microfilaremic Patients before and 12 Mo after Doxycycline Treatment

Plasma concentrations (mean ± SD) of sVEGFR-3 were measured from plasma of microfilaremic patients before and 12 mo after doxycycline treatment (17 doxycycline treated, 16 placebo, see Table 1). The sVEGFR-3 levels decreased significantly at 12 mo (preceding supratesticular lymphatic dilation, see Table 2) in the doxycycline-treated patients (p = 0.0125) to a level close to that of endemic controls whereas there was no difference in the placebo group (paired t-test).

Figure 7. Plasma Levels of VEGF-C of Lymphedema Patients before and 12 Mo after Doxycycline Treatment

Plasma concentrations (mean ± SD) of VEGF-C were measured from plasma of lymphedema patients before and 12 mo after doxycycline treatment. The VEGF-C levels decreased significantly at 12 mo in the doxycycline-treated patients (p = 0.0499), in contrast to the placebo group (paired t-test).

Figure 8. Plasma Levels of sVEGFR-3 of Lymphedema Patients before and 12 Mo after Doxycycline Treatment

Plasma concentrations (mean ± SD) of sVEGFR-3 were measured from plasma of lymphedema patients before and 12 mo after doxycycline treatment. The sVEGFR-3 levels decreased significantly at 12 mo in the doxycycline-treated patients (p = 0.0251) whereas there was no difference in the placebo group (paired t-test).

Figure 9. Grading of the Supratesticular Lymphatic Vessel Dilation of Filarial-Infected Patients Displayed by USG

Dilation of the supratesticular lymphatic vessels was determined by measuring the largest diameter detectable in the two-dimensional b-mode of a portable ultrasound machine. A grading system was developed to determine the degree of lymphatic dilation as follows: (A) category 1: patients with minimal lymphatic dilation of up to 0.2 cm; (B) category 2: patients with mild dilation from 0.21–0.50 cm; (C) category 3: patients with moderate dilation from 0.51–1.0 cm; and (D) category 4: patients with severe dilation of above 1.0 cm.

Figure 10. Lymphedema Stages

The lymphedema stages according to the classification by Dreyer et al. [47]; patients are from this study. (A) Stage 2, swelling that is not reversible overnight. (B) Stage 3, shallow skin folds at the ankle. (C) Stage 4, alteration of skin texture and formation of knobs (arrowheads). (D) Stage 5, presentation of deep skin folds in addition to the alterations of stage 4; (E) Satge 6, presentation of mossy lesion in addition to the alterations of stage 5. (F) Stage 7, inability of patient to perform daily work.