Project description:Lymphogenous metastasis is an important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to the lymphatic vasculature can occur during metastasis, and may aid metastatic spread. We investigated the effect of tumour derived VEGFD on the endothelium of the collecting lymphatic vessels draining primary tumors. We used microarrays to detail the changes in gene expression in the collecting lymphatic endothelium of mice with 293EBNA xenografts compared to 293EBNA xenografts overexpressing VEGFD. Mice were injected with 293EBNA cells (transfected with either empty APEX vector, or vector containing VEGFD) and tumours were allowed to grow to size. Mice were sacrificed and collecting lymphatic vessels were dissected. The endothelial cell population was isolated and RNA was extracted and hybridized on Affymetrix microarrays.
Project description:The muscle cells within the wall of collecting lymphatic vessels exhibit tonic and autonomous phasic contractions, which drive active lymph transport to maintain tissue-fluid homeostasis and support immune surveillance. Damage to the lymphatic muscle cells (LMC) disrupts lymphatic function and is related to various diseases. Despite their importance, knowledge of the transcriptional signatures in LMC and how they relate to lymphatic function in normal and diseases contexts is largely missing. In this study, we have generated to date the most comprehensive transcriptional single-cell atlas—including LMC—of collecting lymphatic vessels in mouse dermis at various ages.
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.
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.
Project description:The goal and objective of this study was to identify the transcriptional profiles differentiating the artery, vein, and lymphatic lineages in the adult rat vasculature with particular emphasis on the unique elements of the collecting lymphatic vessel transcriptome. A 2 x 3 experimental design was utilized in which parallel arteries, veins, and lymphatics from two different tissue beds were examined. The rat thoracic duct was selected as a large, post-nodal collecting lymphatic vessel that exhibits excellent conduit-type behavior while the rat mesenteric lymphatic was selected as a smaller, pre-nodal collecting lymphatic vessel that exhibits excellent pump behavior (see Gashev AA, et al. Microcirculation. 2004 Sep;11(6):477-92. [PMID: 15371129]). The axillary artery and vein were selected for comparison to the thoracic duct due to their similar anatomical position distal to the common junction of the lymphatic and venous vascular trees and represent a large artery and large vein, respectively. The mesentery artery and vein were selected for comparison to the mesenteric lymphatic vessels due to their parallel position within the mesenteric vasculature and represent a small atery and small vein, respectively. A 2 x 3, reference-based, experimental design was utilized consisting of both large (thoracic) and small (mesenteric) arteries, veins, and collecting lymphatic vessels for a total of 6 sample groups with n=6 biological replicates present in each group. All vessels acquired from the same donor animal have the same numerical label and were handled in parallel through all experimental steps. Each vessel sample RNA sample was amplified, labeled with Cy5, and compared to the same Rat Universal Reference RNA sample (Stratagene, La Jolla, CA) that was amplified and labeled with Cy3 dye. No dye swaps were utilized.
Project description:Lymphogenous metastasis is an important event in the progression of many human cancers, and is associated with expression of vascular endothelial growth factor-D (VEGF-D). Changes to the lymphatic vasculature can occur during metastasis, and may aid metastatic spread. We investigated the effect of tumour derived VEGFD on the endothelium of the collecting lymphatic vessels draining primary tumors. We used microarrays to detail the changes in gene expression in the collecting lymphatic endothelium of mice with 293EBNA xenografts compared to 293EBNA xenografts overexpressing VEGFD.
Project description:Afferent lymphatic vessels (LVs) connect peripheral tissues with draining lymph nodes (dLNs) and are important for immune-surveillance and tissue drainage. They begin in the tissue as initial lymphatic capillaries, which are highly permeable and branched vessels specialized in the uptake of macromolecules, fluids and immune cells. Conversely, the downstream collecting LVs are impermeable and contractile structures that transport the taken up lymph and immune cells to the dLN. We and others have recently observed that intralymphatic leukocytes actively migrate within lymphatic capillaries but de-adhere and are passively transported by flow once they have reached in the collecting vessels. Besides potential differences in lymph flow we hypothesize that gene expression differences between capillaries and collectors could account for this transition from a crawling to a flowing mode of migration. In this project we aimed to perform a sequencing-based gene expression analysis of lymphatic endothelial cells (LECs) isolated from lymphatic capillaries and collectors, in order to identify new genes involved in leukocyte migration, as well as genes involved in shaping the morphologic phenotype of capillaries and collectors. For this, murine skin was enzymatically digested and LECs from capillaries or collectors were FACS-sorted and their RNA extracted and subjected to sequencing.
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:Lymphatic valves are specialized units regularly distributed along collecting vessels that allow unidirectional forward propulsion of the lymph, and its efficient transport from tissues to the bloodstream. Lymphatic endothelial cells that cover lymphatic valve sinuses are subjected to complex flow patterns, due to recirculation of the lymph during the collecting vessel pumping cycle. They also express high levels of FOXC2 transcription factor. We used microarrays to study the transcriptional networks controlled by FOXC2 in human lymphatic endothelial cells subjected to oscillatory shear stress or cultured under static conditions.