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.
Accumulation of lipid-laden foam cells in the arterial wall plays a central role in atherosclerotic lesion development, plaque progression, and late-stage complications of atherosclerosis. However, there are still fundamental gaps in our knowledge of the underlying mechanisms leading to foam cell formation in atherosclerotic arteries. Here, we investigated the role of receptor-independent macropinocytosis in arterial lipid accumulation and pathogenesis of atherosclerosis. Genetic inhibition of fluid-phase macropinocytosis in myeloid cells ( LysMCre + Nhe1 fl/fl ) and repurposing of a Food and Drug Administration (FDA)–approved drug that inhibits macrophage macropinocytosis substantially decreased atherosclerotic lesion development in low-density lipoprotein (LDL) receptor–deficient and Apoe −/− mice. Stimulation of macropinocytosis using genetic ( H-RAS G12V ) and physiologically relevant approaches promoted internalization of unmodified native (nLDL) and modified [e.g., acetylated (ac) and oxidized (ox) LDL] lipoproteins in both wild-type and scavenger receptor (SR) knockout ( Cd36 −/− / Sra −/− ) macrophages. Pharmacological inhibition of macropinocytosis in hypercholesterolemic wild-type and Cd36 −/− / Sra −/− mice identified an important role of macropinocytosis in LDL uptake by lesional macrophages and development of atherosclerosis. Furthermore, serial section high-resolution imaging, LDL immunolabeling, and three-dimensional (3D) reconstruction of subendothelial foam cells provide visual evidence of lipid macropinocytosis in both human and murine atherosclerotic arteries. Our findings complement the SR paradigm of atherosclerosis and identify a therapeutic strategy to counter the development of atherosclerosis and cardiovascular disease.
Introduction : Post‐traumatic vasospasm (PTV) is a significant cause of morbidity and mortality following traumatic brain injury (TBI). 1 Delayed PTV is thought to occur due to inflammation from SAH. 1 The risk of symptomatic PTV is associated with the severity of TBI. 1,2,3 Treatment of PTV traditionally involves agents used in aneurysmal vasospasm such as Nimodipine. Intra‐arterial and intravenous verapamil and milrinone have been utilized to treat PTV. 4 We present a rare case of delayed cerebral ischemia caused by PTV without SAH treated with intra‐arterial milrinone and oral verapamil. Methods : This is a case report of a case of a 16‐day delay of cerebral ischemia secondary to PTV. Results : A 19‐year‐old female without significant medical history presented to the emergency room as the restrained driver in a motor vehicle collision involving a car versus a tree. The patient’s Glasgow Coma Scale score was 13 and the initial head CT did not demonstrate SAH with a Rotterdam score of 0. There were multiple fractures and soft tissue contusions noted on imaging which required surgical correction and splinting. The patient’s hospital stay was complicated by a large retroperitoneal hematoma requiring blood transfusions and surgical evacuation. 16 days after admission, the patient experienced sudden‐onset right upper extremity paralysis and weakness of the right leg. CT head demonstrated loss of gray‐white differentiation in the left middle cerebral artery (MCA) territory and CT angiography demonstrated 70% stenosis of the left supra‐clinoid internal carotid artery (ICA) and proximal left MCA. Cerebral angiography demonstrated 60% stenosis left supra‐clinoid ICA and 60% stenosis at the origin of the left MCA consistent with PTV. Left MCA stenosis improved to 20% post 10 mg intra‐arterial milrinone in the left ICA (Figure 1). The patient also received verapamil orally. Transcranial doppler demonstrated elevated peak systolic velocities at 298 cm/s in the left MCA and 276 cm/s in the left ICA. Six days later this improved to 150 cm/s in the left MCA and 151 cm/s in the left ICA. The patient continued to be unable to move her right side against gravity with dense expressive aphasia and dysarthria at discharge to inpatient rehabilitation. At follow‐up 4 weeks later, her right hemiparesis had improved significantly to being able to stand with assistance and her expressive aphasia had improved from one word to occasionally forming several word sentences. Conclusions : PTV is a potentially devastating complication of TBI. As our case demonstrates, the presence of mild TBI and absence of SAH may be falsely reassuring. 2 Additionally, our case report demonstrates that intra‐arterial milrinone causes radiographic improvement in PTV. Further studies are needed for the best screening and diagnostic exams for PTV and therapeutic interventions.
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