Characterization of Atherosclerotic Mice Reveals a Sex-Dependent Susceptibility to Plaque Calcification but No Major Changes in the Lymphatics in the Arterial Wall.

Lymphatics participate in reverse cholesterol transport, and their presence in the arterial wall of the great vessels and prior experimental results suggest their possible role in the development of atherosclerosis. The aim of this study was to characterize the lymphatic vasculature of the arterial wall in atherosclerosis. Tissue sections and tissue-cleared aortas of wild-type mice unveiled significant differences in the density of the arterial lymphatic network throughout the arterial tree. Male and female Ldlr-/- and ApoE-/- mice on a Western diet showed sex-dependent differences in plaque formation and calcification. Female mice on a Western diet developed more calcification of atherosclerotic plaques than males. The lymphatic vessels within the aortic wall of these mice showed no major changes regarding the number of lymphatic junctions and end points or the lymphatic area. However, female mice on a Western diet showed moderate dilation of lymphatic vessels in the abdominal aorta and exhibited indications of increased peripheral lymphatic function, findings that require further studies to understand the role of lymphatics in the arterial wall during the development of atherosclerosis.

CCBE1 regulates the development and prevents the age-dependent regression of meningeal lymphatics.

Recent studies have described the importance of lymphatics in numerous organ-specific physiological and pathological processes. The role of meningeal lymphatics in various neurological and cerebrovascular diseases has been suggested. It has also been shown that these structures develop postnatally and are altered by aging and that the vascular endothelial growth factor C (VEGFC)/ vascular endothelial growth factor receptor 3 (VEGFR3) signaling plays an essential role in the development and maintenance of them. However, the molecular mechanisms governing the development and maintenance of meningeal lymphatics are still poorly characterized. Recent in vitro cell culture-based experiments, and in vivo studies in zebrafish and mouse skin suggest that collagen and calcium binding EGF domains 1 (CCBE1) is involved in the processing of VEGFC. However, the organ-specific role of CCBE1 in developmental lymphangiogenesis and maintenance of lymphatics remains unclear. Here, we aimed to investigate the organ-specific functions of CCBE1 in developmental lymphangiogenesis and maintenance of meningeal lymphatics during aging. We demonstrate that inducible deletion of CCBE1 leads to impaired postnatal development of the meningeal lymphatics and decreased macromolecule drainage to deep cervical lymph nodes. The structural integrity and density of meningeal lymphatics are gradually altered during aging. Furthermore, the meningeal lymphatic structures in adults showed regression after inducible CCBE1 deletion. Collectively, our results indicate the importance of CCBE1-dependent mechanisms not only in the development, but also in the prevention of the age-related regression of meningeal lymphatics. Therefore, targeting CCBE1 may be a good therapeutic strategy to prevent age-related degeneration of meningeal lymphatics.

Ex vivo confocal Raman microspectroscopy of porcine dura mater supported by optical clearing.

The effect of tissue optical clearing (TOC) to increase the probing depth and observe in-depth structure of the ex vivo porcine dura mater was studied by confocal Raman microspectroscopy (CRM). Raman intensities were significantly increased at the depth of 250 µm for all collagen bands after treatment with glycerol. The influence of glycerol on collagen hydration was also investigated. The results indicate that the process of TOC can be divided into three main steps. The first one is a fast process of tissue dehydration accompanied by collagen shrinkage while the second relatively slow process is related to the glycerol penetration into the interfibrillar space of collagen combined with swelling of tissue. The third step is collagen dissociation caused by the high concentration of glycerol. To the best of our knowledge, this study is the first example to introduce the TOC technique in assisting CRM of ex vivo dura mater in-depth probing.

Lymph Flow Induces the Postnatal Formation of Mature and Functional Meningeal Lymphatic Vessels.

Recently, the presence of lymphatics has been demonstrated and characterized in the dura mater, which is in contrast to the well-accepted view indicating the lack of a classical lymphatic drainage system of the central nervous system (CNS). Moreover, the role of meningeal lymphatics in the pathogenesis of Alzheimer's disease and multiple sclerosis was suggested. However, the possible regulators of the developmental program and function of meningeal lymphatics remain unclear. Here, we aimed at characterizing the lymph flow dependence of the developmental program and function of the meningeal lymphatics. First, we demonstrated that lymphatics present in the dura mater are involved in the uptake and transport of macromolecules from the CNS. Meningeal lymphatics develop during the postnatal period which process involves the maturation of the vessels. The formation of mature meningeal lymphatics coincides with the increase of the drainage of macromolecules from the CNS to the deep cervical lymph nodes. Importantly, the structural remodeling and maturation of meningeal lymphatics is impaired in Plcγ2-/- mice with reduced lymph flow. Furthermore, macromolecule uptake and transport by the meningeal lymphatics are also affected in Plcγ2-/- mice. Collectively, lymph flow-induced mechanical forces are required for the postnatal formation of mature and functional meningeal lymphatic vessels. Defining lymph flow-dependence of the development and function of meningeal lymphatics may lead to better understanding of the pathogenesis of neurological diseases including Alzheimer's disease and multiple sclerosis.