The pathologic hallmark of lymphatic filariasis is lymphedema and elephantiasis that has been associated with lymphatic dilatation, tortuosity, and obstruction. It is known, however, that individuals carrying adult worms (with or without microfilaremia) may have lymphangiectasia, acute lymphangitis, and lymphedema of the extremities 
. Further, it has been postulated that with sufficient infection chronicity, individuals who harbor adult Wuchereria bancrofti
will develop lymphangiectasia in the vicinity of the worm nests 
. Although it is not clear what induces the lymphatic dysfunction in filaria-infected individuals, previous studies have shown that filarial antigens do not induce proliferation of HUVEC in vitro
and, in fact, inhibited their proliferation 
. More recent findings, implicate the role of VEGF family members in filarial pathology 
. The availability of markers (Prox-1, podoplanin, LYVE-1, VEGFR-3, among others) to differentiate LEC from blood vascular endothelial cells has facilitated the study of LEC directly. Although LEC have a lower turnover rate compared with blood vascular endothelial cells, on appropriate stimulation in vivo
, LEC are capable of proliferating and migrating to organize into new lymphatic vessels (lymphangiogenesis) 
. That this process occurs in response to filarial infection/antigens is demonstrated by our findings that filarial antigens specifically induce LEC proliferation and/or differentiation and do so in an antigen-specific manner. Compared to the LECs, under identical serum deprived conditions, VEGF-A induced proliferation of the HUVEC was similar to that of filarial antigens. It is not clear if the observed lower proliferative response of the HUVECs (compared to the LEC) to both the filarial antigens and VEGF-A reflects on their maximum proliferative capacity in vitro
. Further, the failure of the filarial antigens to stimulate both the LECs and HUVECs in the presence of serum supplemented media compared to unstimulated controls probably reflects on various growth factors in the sera masking the proliferative capacity of the antigens, which could possibly account for similar observation with HUVECs previously 
. Though VEGF-C (native or the mutant form of VEGFC-156Ser) specifically activates LEC, it was unable to induce significant proliferation of the LEC at the concentration similar to VEGF-A (25ng/ml). Our data show that, unlike the schistosome antigens (SEA or SWAP) that have no effect on LEC (present study) but do induce the proliferation of HUVECS () 
, both adult filarial antigens and serum from patients with lymphatic filariasis induce LEC proliferation. As has been shown by others 
, it is possible that circulating levels of pro-lymphangiogenic factors or circulating parasite antigens (>32,000 Og4C3 units in plasma of infected individuals, data not shown) themselves could be stimulating LECs in vivo
. In addition, MfAg appears to induce lymphatic vessel differentiation and remodeling that may be aided by the alteration of the extracellular matrix.
The lymphangiogenic response requires both upregulation of lymphangiogenic factor expression and downregulation of the inhibitors of lymphangiogenesis 
(typically involved in degradation and synthesis of the extracellular matrix components 
). Although VEGF family members have been considered the prime mediators of lymphangiogenesis, many other mediators have been identified that can also influence the lymphatic vasculature 
. Among these HGF, bFGF, PDGF, angiopoeitins, and IGF-1 are known inducers and/or regulators of lymphangiogenesis; however, we were unable to detect any significant increase in the levels of HGF, b-FGF, ANG-2 or PDGF in the culture supernatants of LEC stimulated with MfAg. This however does not preclude the release of VEGF in in vivo
conditions by the immune cells such as the macrophages that could aid in the filarial pathology. Thus, the host factors mediating lymphangiogenesis and lymphatic differentiation awaits clarification.
Typically, confluent monolayers of lymphatic endothelium in the presence of growth factors form tubular networks that are similar in their structural arrangement to lymphatic capillaries in vivo 
. EM analysis of these tubular structures suggested that the flattened EC form a luminal space comprising one to several EC. Typical studies on lymphangiogenesis utilize collagen- or gelatin-coated culture plates or, for 3-dimensional studies, Matrigel™; however, filarial antigen-induced tube formation did not require any pre-existing matrix, suggesting that laminin and collagen type IV (major components of Matrigel™) are not absolutely essential for differentiation of LEC in vitro
as has been demonstrated in previous in vitro
. This is indicative of the capillary EC inherent capacity to produce and secrete factors that can aid in tissue remodeling (e.g. MMP) 
. The activity of MMP is further controlled by endogenous TIMP that can bind to zymogens or inhibit the activated forms of MMP 
. TIMP-1 and TIMP-2 are the best-known inhibitors that can suppress capillary endothelial cell function in vitro
. The filarial antigen-induced tube formation seen in the present study seemed most dependent on regulation of TIMP-1 and TIMP-2 that were, in turn, associated with increased levels of MMP-1 and MMP-2. Preliminary data suggest that addition of exogenous TIMP-1 and TIMP-2 inhibit the antigen-induced tube formation to a considerable extent (data not shown). It remains to be seen how TIMP and MMP regulate LEC activity upon stimulation with filarial antigens, although their role in development of collateral/accessory lymphatics in cats infected with Brugia pahangi 
and in humans with lymphatic filariasis has been postulated previously.
A characteristic feature of long-term filarial infection in humans and animals is the fibrosis and cellular hyperplasia in and around the lymphatic walls. Infection with the parasites for long periods results in the fibrosis of the infected lymph nodes, which eventually become non-functional and are bypassed by new lymphatic vessels 
. Several animal studies 
have previously shown that EC from infected animals had a decreased number of vesicles (which presumably transport fluid) and an increase in the number of vacuoles (which presumably results from cell damage), suggesting that EC lining the lymphatics inhabited by the filarial worms are affected by the parasites. The injury probably renders these EC less effective in transporting edematous fluid and thereby contributes to the edema and collagen accumulation. Studies on LEC permeability (as an indirect measure of their transport capability) demonstrated that, upon stimulation with filarial antigens, LEC are not rendered hyperpermeable but rather are made less leaky, in large part by the induction of adhesion molecules involved in maintaining cell junctions. A recent study using Dirofilaria immitis
antigen on EC also suggests that antigen-treated monolayers do not differ from the unstimulated controls in the trans-endothelial passage of FITC-Dextran 
. These data suggest that the lymphedema associated with filarial infection is associated with an inability of the parasite-exposed lymphatics to resorb interstitial fluid rather than for fluid to pass through the LEC junctions abnormally. Alternatively, the proinflammatory responses (IL-1, TNF-α) seen in immune effector cells from patients with chronic lymphatic obstruction 
could themselves render the LEC hyperpermeable. The proliferative effects of the adult antigens and more pronounced tube-formation activity of the microfilarial antigens, perhaps is an indication of the lymphatic dilatation/damage seen in the sub-clinical infections. It is not absolutely clear, but the consensus on the progression and development of lymphedema/elephantiasis lies in the death of the parasites that triggers a massive inflammatory response against the parasite products and its endosymbiont wolbachia. By whatever mechanism, the interstitial fluid remains and leads to fibrosis and tissue injury.
Thus, our studies reveal that filarial parasites are capable of inducing lymphangiogenesis in vitro
, a process that is LEC specific and relates to the excretory-secretory components of the filarial parasites and/or the elevated levels of circulating lymphangiogenic factors 
. The new vessels formed appear to be associated with intrinsic extracellular matrix modeling and are not related to expression of prototypical markers of lymphangiogenesis seen in tumor biology. Our data also suggest that filarial lymphedema may not be due to parasite-induced alterations in trans-endothelial transport (as has been hypothesized) but may involve host-expressed inflammatory components. With the elucidation of the stage-specific excretory-secretory components of B. malayi 
, further studies on the effects of these ES products (and their purified components) on the LECs would enable a clearer understanding of the filarial induced lymphatic dysfunction associated with lymphatic filariasis.