Project description:To identify genes regulating the functional specification of collecting lymphatic vessels, we performed transcriptome profiling of dermal ECs isolated from adult mouse ear skin by flow cytometry.
Project description:Tissue lymphatic vessels network plays critical roles in immune surveillance and tissue homeostasis in response to pathogen invasion, but how lymphatic system per se is remolded during infection is less understood. Here, we observed that influenza infection induces a significant increase of lymphatic vessel numbers in the lung, accompanied with extensive proliferation of lymphatic endothelial cells (LECs). Single-cell RNA sequencing illustrated the heterogeneity of LECs, identifying a novel PD-L1+ subpopulation that is present during viral infection but not at steady state. Specific deletion of Pd-l1 in LECs elevated the expansion of lymphatic vessel numbers during viral infection. Together these findings elucidate a dramatic expansion of lung lymphatic network in response to viral infection, and reveal a PD-L1+ LEC subpopulation that potentially modulates lymphatic vessel remolding.
Project description:Kaposi sarcoma is the most common cancer in AIDS patients and is typified by red skin lesions. The disease is caused by the KSHV virus (HHV8) and is recognisable by its distinctive red skin lesions. The lesions are KSHV-infected spindle cells, most commonly the lymphatic endothelial and blood vessel endothelial cells (LEC and BEC), plus surrounding stroma. The effects of KSHV infection of both LEC and BEC were assayed using Affymetrix hgu133plus2 chips at 72 hours post infection.
Project description:The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. While lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control in a bimodal manner the production of extracellular matrix scaffold components and signal ligands critical for lymphatic vessel formation.
Project description:This SuperSeries is composed of the following subset Series: GSE16353: The profile of cellular and KSHV microRNAs in AIDS_KS biopsies (and normal skin control biopsies) GSE16354: Infection of Lymphatic and Blood Vessel Endothelial Cells (LEC and BEC) with KSHV GSE16355: Lymphatic endothelial cells (LEC) transfected with the KSHV microRNA cluster GSE16356: Lymphatic endothelial cells (LEC) treated with a MAF-targeted siRNA Refer to individual Series
Project description:Complex lymphatic anomalies (CLAs) are sporadically occurring diseases caused by the maldevelopment of lymphatic vessels. We and others recently reported that somatic activating mutations in KRAS can cause CLAs. However, the mechanisms by which activating KRAS mutations cause CLAs are poorly understood. Here we show that KRASG12D expression in lymphatic endothelial cells (LECs) during embryonic development impairs the formation of lymphovenous valves and causes the enlargement of lymphatic vessels. We demonstrate that KRASG12D expression in primary human LECs induces cell spindling, proliferation, and migration. It also increases AKT and ERK1/2 phosphorylation and decreases the expression of genes that regulate lymphatic vessel maturation. We show that MEK1/2 inhibition with the FDA-approved drug trametinib suppresses KRASG12D-induced morphological changes, proliferation, and migration. Trametinib also decreases ERK1/2 phosphorylation and increases the expression of genes that regulate the maturation of lymphatic vessels. We also show that trametinib and Cre-mediated expression of a dominant-negative form of MEK1 (Map2k1K97M) suppresses KRASG12D-induced lymphatic vessel hyperplasia in embryos. Last, we demonstrate that conditional knockout of wild-type Kras in LECs does not affect the formation or function of lymphatic vessels. Together, our data indicate that KRAS/MAPK signaling must be tightly regulated during embryonic development for the proper development of lymphatic vessels and further support the testing of MEK1/2 inhibitors for treating CLAs.
Project description:Proliferation and migration of lymphatic endothelial cells (LECs) are essential for lymphatic vessel growth (also known as lymphangiogenesis), which plays a critical role in regulating the tissue fluid balance and immune cell trafficking in physiological and pathological conditions.
Project description:The identification of circulating endothelial progenitor cells has led to speculation regarding their origin as well as their contribution to neovascular development. Two distinct types of endothelium make up the blood and lymphatic vessel system. However, it has yet to be determined whether there are distinct lymphatic-specific circulating endothelial progenitor cells. We isolated circulating endothelial colony forming cells (ECFCs) from whole peripheral blood. These cells are endothelial in nature, as defined by their expression of endothelial markers and their ability to undergo capillary morphogenesis in three-dimensional culture. A subset of isolated colonies express markers of lymphatic endothelium, including VEGFR-3 and Prox-1, with low levels of VEGFR-1, a blood endothelial marker, while the bulk of the isolated cells express high VEGFR-1 levels with low VEGFR-3 and Prox-1 expression. The different isolates have differential responses to VEGF-C, a lymphatic endothelial specific cytokine, strongly suggesting that there are lymphatic specific and blood specific ECFCs. Global analysis of gene expression revealed key differences in the regulation of pathways involved in cellular differentiation between blood and lymphatic-specific ECFCs. These data indicate that there are two distinguishable circulating ECFC types, blood and lymphatic, which are likely to have discrete functions during neovascularization. RNA was isolated from 2 blood-specific ECFC cell lines and 2 lymphatic-specific ECFC cell lines 3 separate times each
Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.