We used a whole-slide imaging system to examine the distribution and alteration of lymphatic capillaries and mature lymphatic vessels in joint and long bone sections from normal mice and mice with chronic arthritis including OA and RA. We found that both lymphatic capillaries and mature vessels were present in the various soft tissues of normal joints and in the periosteum of long bones. Lymphatic vessels were located mainly in the loose connective and adipose tissues adjacent to the synovial membrane within the joint capsule. The areas of the lymphatic capillaries were increased significantly in joints with mild arthritis. Areas of the lymphatic vessels, mainly mature lymphatic vessels, were decreased markedly in joints with severe arthritis in both OA and RA. VEGF-C increased lymphatic vessel maturation in RA joints. Our findings suggest that the lymphatic system may play a critical role in arthritis pathogenesis and therapy, and these factors require further investigation. Our findings also indicate that the whole-slide imaging system is an excellent tool for investigating lymphatic vasculature in hard tissues.
The presence of numerous lymphatic vessels in the periosteum is particularly interesting. Periosteum plays a critical role in bone repair by providing blood supply and mesenchymal stem cells (Schonmeyr et al. 2009
, Zhang et al. 2005
). It is not known, however, whether lymphatic-mediated events also contribute to bone repair. Because sufficient lymphatic drainage helps resolve inflammation, and because the inflammatory reaction is one of the key components of bone repair, it is conceivable that the lymphatic system plays a role in this process. This possibility must be examined in the future. The IHC staining method did not detect lymphatic vessels in bone marrow (Edwards et al. 2008
, Zhang et al. 2008a
), which indicates that fluid clearance in the marrow cavities is by venous sinusoids rather than lymphatics. With the help of the VS-ASW FL system, we occasionally (~ 10% of samples) observed lymphatic vessels in bone cortex and marrow cavities (data not shown). The exact role of these vessels in bone metabolism, however, is not clear at this time.
OA is a degenerative disease of the joints that is associated with high levels of catalytic enzymes and their products in the articular space. IHC staining of joint sections with anti-LYVE-1 or podoplanin antibodies indicated that the number of lymphatic vessels was increased (Xu et al. 2003
) or decreased (Walsh et al. 2012
) in OA patients. The discrepancy may be due to the sampling site and/or different patient populations. Capillary and mature lymphatic vessels were not distinguished in these descriptive studies. We found markedly decreased cartilage and mature lymphatic vessels in OA mice fed a high fat diet (). There are two possible explanations for this. One is that the pathological process associated with OA progression inhibits lymphatic vessel maturation, as seen in RA joints. Another possibility is that high fat diet influences maturation of lymphatic vessels. It was reported recently that hypercholesterolemic mice exhibit decreased smooth muscle cell coverage [Q6]
and decreased lymphatic transport of fluid (Lim et al. 2009
). In humans, a strong correlation has been shown between type 2 diabetes and OA (Holliday et al. 2011
). Our earlier study also indicated that a high fat diet leads to weight gain, diabetes and progression of OA (Mooney et al. 2011
). Thus, the possibility that a high fat diet accelerates OA progression by hindering the maturation of lymphatic capillaries in OA joints must be investigated in the future. In particular, investigation of whether a high fat diet affects lymphatic drainage function is required.
RA is an inflammatory erosive disease associated with pain, swelling and stiffness of affected joints (Bergman 2006
). We reported earlier that lymphatic drainage is associated with the onset, progression and flare-up of RA (Li et al. 2010
) and that lymphatic growth factor, VEGF-C, promotes resolution of inflammation in RA joints by promoting lymphatic vessel formation and lymph drainage (Zhou et al. 2011
). In our earlier studies, however, we did not distinguish capillaries from mature lymphatic vessels. We do not know whether the severity of RA is related to impaired lymphatic vessel maturation or whether it is affected by VEGF-C. This is important, because many studies have demonstrated that inflammation induces lymphangiogenesis, but that inflammation-induced lymphatic vessels are not sufficiently functional. We found that mature lymphatic vessels were observed rarely in the inflammatory pannus of TNF-Tg mice and that VEGF-C treatment significantly increased the areas of capillary and mature lymphatic vessels, which suggests that VEGF-C not only stimulates lymphangiogenesis, but also lymphatic vessel maturation. This raises the possibility of using VEGF-C as a new lymphatic therapy for patients with arthritis.
The whole-slide imaging is an excellent tool for studying lymphatics in hard tissues. With this new tool, we have begun to explore the presence, distribution, and alteration of lymphatic capillaries and mature vessels in mouse models of arthritis, which may lead to the development of new mechanisms and treatments for patients with arthritis.