Tissue responses in inflammatory arthritis include a number of complex pathological changes in affected joints, including edema. During inflammatory reactions, factors that increase the drainage function of lymphatic vessels could lead to enhanced clearance of inflammatory cells and pro-inflammatory cytokines from tissues to draining LNs, resulting in less severe inflammation. Here, we report for the first time using TNF-Tg mice as a model of chronic inflammatory arthritis, that promotion of lymphangiogenesis by intra-articular injection of AAV-VEGF-C decreased the severity of the synovitis and joint destruction accompanied by increased numbers of lymphatic vessels and improved lymph flow from the affected joint area to draining lymph nodes. These findings suggest that VEGF-C treatment can both affect the severity of inflammatory process and improve the function of new lymphatics.
It has been a general accepted concept that inflammation stimulates lymphangiogenesis as a compensatory mechanism to enhance the clearance of inflammatory products. This conclusion is mainly based on immunostaining observations of inflamed tissues with antibodies specifically recognizing lymphatic endothelium cells, such as LYVE-1 (2
). Increase in lymphangiogenesis is due to inflammatory cells such as macrophages and myeloid precursors producing lymphatic growth factors (2
), leading to proliferation of lymphatic endothelium cells and perhaps remodeling of existing lymphatic networks. For instance, we and others have reported that TNF stimulates VEGF-C production by synovial fibroblasts (41
) and macrophages (21
). However if these newly formed lymphatic vessels in response to inflammatory stimuli fully function is not clear. Our findings suggest that increased LYVE-1+ lymphatics vessels in synovium of TNF-Tg mice are either functionally impaired or present in insufficient numbers to be fully effective and that AAV-VEGF-C can rectify these deficiencies. This finding is consistent with a recent report using keratin 14-VEGF-A and VEGF-C double transgenic mice, in which ICG lymphatic imaging indicates that chronic skin inflammation induced by VEGF-A transgene is associated with impaired lymphatic drainage from inflamed sites to local draining LNs. Over-expressing VEGF-C under the same promoter reduces the degree of inflammation and improves the lymph flow (42
). Thus functional improvement of lymphatic vessels within the inflammatory sites and surrounding tissues is another therapeutic alternative to inflammation-induced edema and tissue damage.
The roles of vascular growth factors and their receptors in lymphangiogenesis in inflammatory conditions have only recently begun to be elucidated. VEGF-A binds to VEGFR-2, while VEGF-C and D bind to VEGFR-3 on lymphatic endothelium to regulate lymphatic vessel formation in physiologic conditions. However, it is not clear if one of these vascular growth factors or receptors plays a dominant role in the induction of new lymphatic vessel formation during inflammation. Most studies have focused on VEGF-A because of its clearly defined role in new blood vessel formation in many inflammatory processes. For example, in UVB-induced inflammation, UVB up-regulates epidermal VEGF-A expression resulting in hyper-permeability of cutaneous lymphatic vessels and decreased lymphatic drainage. In this model, blockade of VEGF-A reduced inflammation and the size of lymphatic vessels, while VEGF-A over-expression increased the number of lymphatic vessels at the inflammatory site as well as in draining lymph nodes (42
). Similarly, in TNF-Tg arthritic mice, blockade of VEGF-A signaling through a VEGFR-2 neutralizing antibody diminished synovial inflammation as well as the size of lymphatic vessels in joints and draining lymph nodes (15
The role of VEGF-C in inflammation has not been clear to date. In chronic airway inflammation induced by Mycoplasma pulmonis
) or in chronic arthritis induced by TNF over-expression (15
), systemic blockade of VEGFR-3 signaling caused exaggerated tissue edema and damage. These findings suggest that VEGF-C-mediated activation of the VEGFR-3 pathway in lymphatic endothelium might possibly play an important role in counteracting the pro-inflammatory effects of VEGF-A that are mediated by VEGFR-2. To support this hypothesis, dermal administration of VEGF-C that specifically activates VEGFR-3 but not VEGFR-2 reduces inflammation-induced skin edema (1
). In current study, we demonstrated that intra-articular injection of VEGF-C reduces the degree of tissue damage and bone destruction and improves lymph flow in joints, highlighting the importance of lymphatic flow. However, does VEGF-C only act on lymphangiogenesis or does it possibly have chemotactic or other immunomodulatory effects? VEGF-C increases survival of lymphatic endothelium, sprouting of lymphatic vessels, chemotactis of monocytes, and migration and invasion of cancer cells. A recent study indicated that VEGF-C blockade reduces CD4+ T cell infiltration in a mouse cardiac allografts model (44
) due to inhibition of lymphatic vessel formation. However, there is no report describing that VEGF-C has immunomodulatory effects. Thus if VEGF-C works through other mechanisms to reduce joint inflammation apart from improvement of lymphatic drainage should be investigated in the future, such as whether VEGF-C stimulates its targeting cells to produce inflammatory inhibitors.
Currently, we do not fully understand the molecular mechanisms mediating lymphangiogenesis in RA joints, especially why newly formed lymphatic vessels are not fully functional. We speculate that observed lymphatic vessels in the tissue sections of RA synovium are lymphatic capillaries, which can be identified by LYVE-1. However, these IHC results cannot detect mature lymphatic vessels, which are LYVE-1 negative, have valves and execute the lymph transport function. Development of tools to identify these matures lymphatic vessels morphologically in joint synovium are essential for assessing changes of lymphatics during RA development and progression. Finally, although locally administered VEGF-C reduced the severity of inflammation and joint destruction and improved lymphatic drainage, the effects were incomplete, suggesting that either the concentration of VEGF-C attained locally was insufficient or more likely that other factors contribute to the inflammation, joint destruction and lymphatic drainage. Thus it may be necessary to use combinations of therapies to achieve optimal responses.
In summary, our study and others using different models of inflammation clearly demonstrate that lymphangiogenesis developing in response to inflammation has a limited compensatory effect overall on resolution of the process (2
). These inflammation-induced lymphatic vessels are not sufficiently functional, but their function can be improved through the administration of exogenous VEGF-C or other lymphatic factors. Further studies will be required to determine the precise nature of the defect in inflammation-induced lymphatic vessels, if this is cell-mediated and if factors produced during inflammation inhibit lymphatic function.