Project description:The lymphatic vascular system maintains tissue fluid homeostasis, helps mediate afferent immune responses and promotes cancer metastasis. MicroRNAs (miRNAs) have recently emerged as key and potent regulators of the genome that control virtually all aspects of cell and organism biology. Surprisingly, the physiological importance and functional activities of miRNAs in the lymphatic vascular system have not been explored. To address this, we first defined the in vitro miRNA expression profiles of primary human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BVECs). Comparative analysis of these profiles identified 4 BVEC-signature and 2 LEC-signature miRNAs. Further expression analysis by quantitative real-time PCR analysis and by in situ hybridization (ISH) studies confirmed these vascular lineage-specific expression patterns in vivo. Functional characterization of the BVEC-signature miRNA, miR-31, identified a novel BVEC-specific post-transcriptional regulatory mechanism that inhibits lymphatic-specific transcription programs in vitro and lymphatic vascular development during Xenopus embryogenesis. These effects are, in part, mediated via direct post-transcriptional repression of PROX1, a master regulator of lymphatic lineage-specific differentiation. Together, these findings indicate that miR-31, and miRNAs in general, are potent regulators of vascular lineage-specific differentiation and development. 500,000 LECs were transfected with 2 ÂµM Pre-miR-31 or Pre-miR-Neg molecules in biological duplicate and total RNA isolated using the mirVana isolation kit 48 hours post-transfection. The transcriptome profiles of these cells were defined using the Applied Biosystems Human Genome Survey Microarray v2.0 as described. Briefly, dioxigenin-UTP-labeled cRNA was generated from 1.5 Âµg of total RNA using the NanoAmp RT-IVT Labeling Kit (Applied Biosystems). 20 Âµg cRNA were fragmented and hybridized to the microarrays using the Applied Biosystems Chemiluminescence Detection Kit. Signal detection, image acquisition and initial analyses were performed using the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer. Raw data were normalized using Quantile normalization available from R/Bioconductor. Present calls were defined based on average signal-to-noise ratios (S/N ratio) >3 and quality (error) values <5,00025. Feature signal intensities were converted to log2 values. miR-31-repressed genes were identified based on present calls in both Pre-miR-Neg arrays, log2(Pre31/PreNeg) â?¤-0.59 and p-values <0.05, while miR-31-induced genes were present in both Pre-miR-31 arrays, had log2(Pre31/PreNeg) â?¥0.59 and p-values <0.05. P-values were calculated using empirical Bayes statistics for differential expression.

Project description:Angiogenesis and lymphangiogenesis have important roles in cancer progression and chronic inflammatory diseases, but efficient therapies against these diseases have been hampered by the lack of identified vascular lineage-specific markers and growth factors. Using transcriptional profiling of matched pairs of human dermal blood vascular and lymphatic endothelial cells, we first identified 236 lymphatic and 342 blood vascular signature genes. In silico analyses of the biologic pathways associated with these genes revealed lineage-specific functions for each cell type. Using a selection of 85 identified vascular lineage-specific genes, we developed a TaqMan RT-PCR-based, microfluidic card-formatted low-density microvascular differentiation array (LD-MDA) that was used to reliably identify and quantify the degree of lineage-specific differentiation in different types of endothelial cells, and to detect admixture of lymphatic endothelial cells in commercial preparations of microvascular endothelial cells. Application of Prediction Relevance Ranking and analysis of variance of LD-MDA expression profiles of 43 lesional skin samples obtained from patients with the chronic inflammatory disease psoriasis led to identification of cytokines which are significantly associated with angiogenesis or lymphangiogenesis in vivo. In particular, interleukin-7 and fibroblast growth factor-12 were identified as novel (lymph)angiogenic factors. This technology provides a novel tool to quantify lineage-specific vascular differentiation and to characterize (lymph)angiogenesis in clinical samples obtained from angiogenic diseases. Keywords: Quantitative real time RT-PCR, psoriasis (chronic inflammation) study Guided by the gene array results, we selected 54 LEC-specific genes and 31 BEC-specific genes, based upon their consistent and strong specific expression in LEC or BEC, as well as on their assignment to important biological pathways. In addition, the five pan-endothelial cell marker genes PECAM-1, vWF, KDR, TEK, CDH5 and the six endogenous control genes ACTB, GAPDH, PGK1, PPIA, RPLP0 and S18 were included in the design of the LD-MDA. After extraction of total RNA, the mRNA expression levels of the 96 genes were analyzed by quantitative RT-PCR using the 7900HT Real-Time PCR System (Applied Biosystems).

Project description:Angiogenesis and lymphangiogenesis have important roles in cancer progression and chronic inflammatory diseases, but efficient therapies against these diseases have been hampered by the lack of identified vascular lineage-specific markers and growth factors. Using transcriptional profiling of matched pairs of human dermal blood vascular and lymphatic endothelial cells, we first identified 236 lymphatic and 342 blood vascular signature genes. In silico analyses of the biologic pathways associated with these genes revealed lineage-specific functions for each cell type. Using a selection of 85 identified vascular lineage-specific genes, we developed a TaqMan RT-PCR-based, microfluidic card-formatted low-density microvascular differentiation array (LD-MDA) that was used to reliably identify and quantify the degree of lineage-specific differentiation in different types of endothelial cells, and to detect admixture of lymphatic endothelial cells in commercial preparations of microvascular endothelial cells. Application of Prediction Relevance Ranking and analysis of variance of LD-MDA expression profiles of 43 lesional skin samples obtained from patients with the chronic inflammatory disease psoriasis led to identification of cytokines which are significantly associated with angiogenesis or lymphangiogenesis in vivo. In particular, interleukin-7 and fibroblast growth factor-12 were identified as novel (lymph)angiogenic factors. This technology provides a novel tool to quantify lineage-specific vascular differentiation and to characterize (lymph)angiogenesis in clinical samples obtained from angiogenic diseases. Keywords: Quantitative real time RT-PCR, cell type comparison Guided by the gene array results, we selected 54 LEC-specific genes and 31 BEC-specific genes, based upon their consistent and strong specific expression in LEC or BEC, as well as on their assignment to important biological pathways. In addition, the five pan-endothelial cell marker genes PECAM-1, vWF, KDR, TEK, CDH5 and the six endogenous control genes ACTB, GAPDH, PGK1, PPIA, RPLP0 and S18 were included in the design of the LD-MDA. The LD-MDA was then used to evaluate the lineage-specific differentiation of a total of 10 independent lines of primary human dermal LEC, of 8 independent lines of primary human dermal BEC, of two independent lines of HUVEC cells, of the immortalized human microvascular endothelial cell line HMEC-1, of the immortalized human epidermal keratinocyte line HaCaT, and of primary human dermal fibroblasts. After extraction of total RNA, the mRNA expression levels of the 96 genes were analyzed by quantitative RT-PCR using the 7900HT Real-Time PCR System (Applied Biosystems).

Project description:ERG and FLI1 are important regulators of angiogenesis, but their role in lymphatic vasculature is less known. The goal of this study was to determine the role of ERG and FLI1 in postnatal lymphangiogenesis and their involvement in SSc lymphatic system defects. IF were used to detect ERG and FLI1 in SSc and healthy controls (HC) skin biopsies. Human lymphatic endothelial cells (LECs) were used in cell culture experiments. Transcriptional and chromatin analysis of ERG or FLI1 silenced LECs was performed using microarrays and ATAC-seq, respectively. Effects of ERG and FLI1 deficiency on in vitro tubulogenesis was examined using Matrigel assay. Erg and Fli1 endothelial specific knockouts (ErgCKO and Fli1CKO) and lymphatic specific knockouts (ProxErgCKO and ProxFli1CKO) were generated to examine vessel regeneration. ERG and FLI1 protein levels were reduced in blood and lymphatic vasculature in SSc skin biopsies. ERG and FLI1 were shown to regulate genes involved in lymphatic vessel specification, including VEGFR3/FLT4, LYVE-1, and PROX1, and this effect was, at least in part, due to chromatin remodeling. ERG/FLT4 pathway regulated in vitro tubulogenesis in LECs. Deficiency of Erg and Fli1 impaired function of blood and lymphatic vessel during wound healing. ERG and FLI1 are essential regulators of blood and lymphatic vessel regeneration. Deficiency of ERG and FLI1 in SSc endothelial cells, may contribute to rarefaction of blood and lymphatic vasculature in SSc patients. Microarray analysis was performed at The Boston University Microarray Core Facility. Affymetrix CEL files were normalized to produce gene-level expression values using the implementation of the Robust Multiarray Average (RMA) in the affy package (version 1.36.1) included within in the Bioconductor software suite (version 2.12) and an Entrez Gene-specific probe set mapping from BrainArray (version 16.0.0). RLE and NUSE quality metrics were computed using the affy PLM Bioconductor package (version 1.34.0). All microarray analyses were performed using the R environment for statistical computing (version 2.15.1).

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Homo sapiens

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