Decreased lymphangiogenesis in mice with osteoarthritis under high fat diet

Abstract

Purpose: We have reported that lymphatic drainage plays a critical role in limiting the severity of joint tissue injury in mouse model of rheumatoid arthritis by removing catalytic factors and inflammatory cells from interstitial fluids to the local draining lymph nodes. However the implication of lymphatic in the pathogenesis of osteoarthritis (OA) is un-known, especially in OA with diabetic because patients with diabetic often have abnormal micro-vasculature. Currently, there is no method to assess lymphatic draining function in mouse knee as an outcome measure to study lymphatic in OA. Here we examined lymphatic vessels in joint sections from a mouse model of OA with diabetes, and developed a near-infrared lymphatic image technique to monitor lymphatic drainage from knee to local draining lymph nodes in real time. Methods: OA was initiated by a meniscall-ligamentous injury (MLI) procedure in the right knee joint. Type 2 diabetic was induced by feeding mice with high fat (HF, 60% kcal) diet. Controls included mice with sham-operation in the left knee and fed by low fat diet (LF, 10% kcal). 5 wks-old C57/BL/6 mice were placed on HF or LF for 8 wks and then received MLI or sham-operation to initiate OA. Mice were sacrificed 16 wks after the MLI. The knee joints were subjected to histology and IHC stain with anti-podoplanin antibody to identify lymphatic vessels and anti-alpha smooth muscle actin (aSM) antibody to identify aSM covered vessels. A group of 12-wks-old mice were received intra-articular injection of the infrared dye, indocyanine green (ICG), the path and signal intensity of ICG in the knee were recorded under an infrared lymphatic imaging system at 1, 2, 4, 6, and 24 hrs. The ICG clearance was calculated. Results: MLI induced OA-like changes in right knee joints, including increased Chamber score, decreased cartilage thickness and osteophyte formation, which was more severe in the HF group. IHC detected that podoplanin+ vessels had thin wall with large lumen, a feature of lymphatic vessels. These podoplanin+ lymphatic vessels were localized in the connective tissue around joint capsules and periosteam. aSM+ vessels appeared to have thick wall and did not overlap with the podoplanin+ lymphatic vessels, suggesting that they are likely small blood vessels. Interestingly, the density of lymphatic and blood vessels were similar among sham-LF, OA-LF, and sham-HF groups, but remarkably decreased in OA-HF group. Decreased podoplanin+ vessels were more obvious than those of aSM+ vessels. Lymphatic imaging revealed that lymph drains from knee tissues to iliac lymph nodes via a single collecting lymphatic vessel. No lymphatic pulse was observed. The ICG signal intensity declined rapidly after injection. The ICG clearance (vs time 0) was 15% at 2 hrs, 60% at 4 hrs, 80% at 6 hrs and 100% at 24 hrs, indicating an efficient lymphatic drainage in WT mice. Conclusions: HF diet accelerates the progression of OA in a type 2 diabetic mouse model, which is associated with decreased lymphatic vessel formation. The near-infrared lymphatic image can be used to monitor lymphatic draining function in mouse knees longitudinally. The involvement of lymphatic in OA pathology is currently under investigation.