Real-time quantitative PCR analysis of human collecting lymphatic vessels
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ABSTRACT:
PROVIDER: PRJNA643798 | ENA |
REPOSITORIES: ENA
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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.
2012-01-15 | E-GEOD-34135 | biostudies-arrayexpress
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.
2012-01-15 | GSE34135 | GEO
Project description:The aim of this feasibility study was to assess whether inflammatory status of the lymphatic vessels (LVs) and/or changes in the miRNA profile of the LVs have potential prognostic and predictive value for overall outcome and risk of relapse. Tissue specimens were obtained from the external iliac vessels draining the pelvic region of patients undergoing debulking surgery (n=10). The tissue was transported directly from surgical theatre to the laboratory in ice-cold sterile Phosphate Buffered Saline. LVs were identified and isolated along with a small amount of surrounding tissue under a dissection microscope within a sterile laminar flow cabinet. Each vessel was divided into two sections. One half of each vessel was further cleaned of surrounding fatty tissue, placed in RNA stabilising solution, frozen on dry ice and stored at -20°C. The miRNAs were extracted with an miRNA easy kit and reverse-transcribed to cDNA using a miScript II RT Kit . Subsequent qPCR was carried out using a miScriptSybrGreen real-time PCR kit and pre-defined miScript miRNA PCR Array. Quantification of the inflammatory state (low, medium and high) and presence of cancer-infiltration of each LV was carried out using immunohistochemistry with the remaining half of each vessel. The LV miRNA expression profiling was then analysed in the context of high versus low inflammation, and cancer-infiltrated versus non-cancer-infiltrated. Results were correlated with clinical outcome data including relapse with an average follow-up time of 13.3 months.
2020-07-03 | GSE153719 | GEO
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.
2012-06-16 | GSE22034 | GEO
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.
2012-06-15 | E-GEOD-22034 | biostudies-arrayexpress
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.
2012-06-22 | E-GEOD-17015 | biostudies-arrayexpress
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.
2021-03-30 | GSE159842 | GEO
Project description:Generation of specialized blood vessels via lymphatic transdifferentiation
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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.
2021-05-07 | E-MTAB-9175 | biostudies-arrayexpress
Project description: Not available
| S-EPMC6961945 | biostudies-literature