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Atherosclerosis is a progressive arterial disease arising from imbalanced lipid metabolism and a maladaptive immune response. The lymphatic system ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells to draining lymph nodes, thereby potentially affecting atherogenesis. Endothelial cell-specific deletion of Pannexin1 (Panx1) in apolipoprotein E-deficient (Apoe-/-) mice increased atherosclerosis, suggesting a protective role for Panx1 channels in arterial endothelial function. Here, we investigated the role of Panx1 in lymphatic endothelial cells (LECs) in the initiation and the progression of atherosclerosis. Male or female Prox1-CreERT2+Panx1fl/flApoe-/- and Panx1fl/flApoe-/- mice were fed a high cholesterol diet (HCD) for 6 or 10 weeks. Tamoxifen-induced deletion of Panx1 was performed before or after 4 weeks of HCD. Body weight and serum lipid profiles were determined. The atherosclerotic plaque burden was assessed by Sudan-IV staining on thoracic-abdominal aortas and in aortic roots. Plaque composition was determined by immunohistochemistry. No differences in serum cholesterol, LDL and HDL were observed between genotypes and between sexes after HCD. Bodyweight, serum triglycerides and free fatty acid levels were higher before and after 6 weeks of HCD in male Prox1-CreERT2+Panx1fl/flApoe-/- and control Panx1fl/flApoe-/- mice compared to females of the same genotypes, which was associated with more lipids and inflammatory cells in their atherosclerotic plaques. In contrast, the atherosclerotic plaque burden was higher in female mice. The progression of atherosclerosis in male mice was not different between genotypes. However, female Prox1-CreERT2+Panx1fl/flApoe-/- mice showed enhanced progression of atherosclerosis compared to Panx1fl/flApoe-/- controls of the same sex. In addition, atherosclerotic lesions in female, but not in male, Prox1-CreERT2+Panx1fl/flApoe-/- mice showed T cell enrichment. Altogether, our results reveal differential sex-dependent effects of Panx1 in lymphatic endothelium on the progression of atherosclerosis. [ABSTRACT FROM AUTHOR]

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Pannexin1 (Panx1) ATP channels are important in adipocyte biology, potentially influencing energy storage and expenditure. We compared the metabolic phenotype of young (14 weeks old) and mature (20 weeks old) wild-type (WT) and Panx1−/− mice exposed or not to cold (6 °C) during 28 days, a condition promoting adipocyte browning. Young Panx1−/− mice weighed less and exhibited increased fat mass, improved glucose tolerance, and lower insulin sensitivity than WT mice. Their energy expenditure and respiratory exchange ratio (RER) were increased, and their fatty acid oxidation decreased. These metabolic effects were no longer observed in mature Panx1−/− mice. The exposure of mature mice to cold exacerbated their younger metabolic phenotype. The white adipose tissue (WAT) of cold-exposed Panx1−/− mice contained more small-sized adipocytes, but, in contrast to WT mice, white adipocytes did not increase their expression of Ucp1 nor of other markers of browning adipocytes. Interestingly, Glut4 expression was already enhanced in the WAT of young Panx1−/− mice kept at 22 °C as compared to WT mice. Thus, Panx1 deletion exerts overall beneficial metabolic effects in mice that are pre-adapted to chronic cold exposure. Panx1−/− mice show morphological characteristics of WAT browning, which are exacerbated upon cold exposure, an effect that appears to be associated with Ucp1-independent thermogenesis. [ABSTRACT FROM AUTHOR]

The lymphatic network of capillaries and collecting vessels ensures tissue fluid homeostasis, absorption of dietary fats and trafficking of immune cells. Pannexin1 (Panx1) channels allow for the passage of ions and small metabolites between the cytosol and extracellular environment. Panx1 channels regulate the pathophysiological function of several tissues in a sex‐dependent manner. Here, we studied the role of Panx1 in lymphatic function, and potential sex‐dependent differences therein, in Prox1‐CreERT2Panx1fl/fl and Panx1fl/fl control mice. Panx1 expression was higher in lymphatic endothelial cells (LECs) of male mice. Lymphatic vessel morphology was not affected in Prox1‐CreERT2Panx1fl/fl male and female mice. Lymphatic drainage was decreased by 25% in male Prox1‐CreERT2Panx1fl/fl mice, but was similar in females of both genotypes. Accordingly, only male Prox1‐CreERT2Panx1fl/fl mice exhibited tail swelling, pointing to interstitial fluid accumulation in males upon Panx1 deletion in LECs. Moreover, serum triglyceride and free fatty acid levels raised less in Prox1‐CreERT2Panx1fl/fl mice of both sexes in an oral lipid tolerance test. Finally, the percentage of migratory dendritic cells arriving in draining lymph nodes was increased in Prox1‐CreERT2Panx1fl/fl female mice, but was comparable between male mice of both genotypes. Our results point to a LEC‐specific role for Panx1 in the functions of the lymphatic system. [ABSTRACT FROM AUTHOR]

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease., Introduction Active and complex signaling is involved both in the growth of new vessels and their stabilization and maintenance. Blood vessel stabilization encompasses proliferation arrest, restoration of the vascular barrier [...]

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 ([Cx40.sup.-/-]). In vivo, hypoxemia was more severe in [Cx40.sup.-/-] mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not [Cx40.sup.-/-], mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype [Ca.sup.2+] channels, cytosolic phospholipase [A.sub.2], and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner., Introduction Hypoxic pulmonary vasoconstriction (HPV) is a fundamental physiological mechanism by which the lung optimizes ventilation/perfusion (V/Q) matching, redirecting blood flow from poorly to better ventilated areas (1). Yet in [...]