Role of R-spondin 2 in arterial lymphangiogenesis and atherosclerosis

Abstract

Background Impaired lymphatic drainage of the arterial wall results in intimal lipid accumulation and atherosclerosis. However, the mechanisms regulating lymphangiogenesis in atherosclerotic arteries are not well understood. Our studies identified elevated levels of matrix protein R-Spondin 2 (RSPO2) in atherosclerotic arteries. In this study, we investigated the role of RSPO2 in lymphangiogenesis, arterial cholesterol efflux into lesion-draining lymph nodes and development of atherosclerosis. Methods and results The effect of RSPO2 on lymphangiogenesis was investigated using human lymphatic endothelial cells in vitro and implanted Matrigel plugs in vivo. Cellular and molecular approaches, pharmacological agents, and siRNA silencing of RSPO2 receptor LGR4 were used to investigate RSPO2-mediated signaling in lymphatic endothelial cells. In vivo LDL tracking and perivascular blockade of RSPO2-LGR4 signaling using LGR4-ECD pluronic gel in hypercholesterolemic mice were utilized to investigate the role of RSPO2 in arterial reverse cholesterol transport and atherosclerosis. Immunoblotting and imaging experiments demonstrated increased RSPO2 expression in human and mouse atherosclerotic arteries compared to non-atherosclerotic controls. RSPO2 treatment inhibited lymphangiogenesis both in vitro and in vivo. LGR4 silencing and inhibition of RSPO2-LGR4 signaling abrogated RSPO2-induced inhibition of lymphangiogenesis. Mechanistically, we found that RSPO2 inhibits PI3K-AKT-eNOS signaling via LGR4 and inhibits activation of the canonical Wnt-β-catenin pathway. ApoE-/- mice treated with LGR4-ECD developed significantly less atherosclerosis compared with control treatment. Finally, increased arterial lymphatic vessel density and improved lymphatic drainage of fluorescently-labeled LDL to deep cervical lymph nodes were observed in LGR4-ECD-treated mice. Conclusions These findings demonstrate that RSPO2 inhibits lymphangiogenesis via LGR4 and downstream impairment of AKT-eNOS-NO signaling. These results may also inform new therapeutic strategies to promote lymphangiogenesis and improve cholesterol efflux from atherosclerotic arteries. Translational Perspective Atherosclerotic cardiovascular disease is the leading cause of death worldwide. Thus, attenuation of atherosclerotic lesion formation and prevention of its cardiovascular complications is an urgent medical need. The findings of the present study that inhibition of LGR4-mediated signaling increases arterial lymphangiogenesis, improves lymphatic drainage from the vessel wall and attenuates atherosclerosis, provide a framework from which novel therapeutic strategies to augment lymphatic vessel density and reduce atherosclerotic lesion formation can be developed and used for the treatment of patients with atherosclerosis. This pathway may also have important implications in other pathological conditions associated with lymphatic dysfunction, such as lymphedema, obesity, hypertension, and impaired wound healing.

Oxidatively Modified LDL Suppresses Lymphangiogenesis via CD36 Signaling

“…Previous studies demonstrated that lymphatic vessels are primarily expressed in the adventitial layer of large and medium-sized arteries and disruption of lymphatic drainage promotes atherosclerotic lesion formation [ 4 , 25 , 30 ]. Therefore, we next examined whether oxLDL treatment regulates lymphangiogenesis in vitro and in vivo using a Matrigel-based tube formation assay and Matrigel plug assay, respectively.…”

“…Previous studies have demonstrated the role of oxidative stress in the suppression of lymphangiogenesis [ 25 , 27 ]. Therefore, we examined whether oxLDL treatment induces intracellular reactive oxygen species (ROS) production in LEC using the H2DCFDA fluorescence method.…”

“…The presence or absence of atherosclerotic lesions was confirmed by Joseph White, Director of Autopsy Services at Augusta University and Oil Red O staining. Additional information about the tissue donors, including cause of death, comorbidities, and race is available in our previous publication [ 25 ]. The study was approved by the Biological Safety Office, Augusta University (BSP# 1458) and conducted in accordance with the guidelines in the Declaration of Helsinki.…”

“…Lipid deposition in human aortic sections was determined using Oil Red O staining as previously described [ 25 ]. Briefly, harvested tissue was fixed in 4% PFA for 48 h, embedded in OCT compound and 8 µm sections prepared.…”

“…In vitro lymphangiogenesis was determined using a Matrigel tube formation assay as previously described [ 25 ]. Briefly, LEC (20,000 cells/well) in basal medium MV 2 (PromoCell GmbH, 0.5% FBS) containing vehicle (PBS), nLDL (50 or 100 µg/mL) or oxLDL (50 or 100 µg/mL) were seeded onto solidified Matrigels in wells of a 96-well plate and incubated for 6 h at 37 °C in a humidified incubator.…”