primary human dermal microvascular lecs  (Lonza)

Journal: The Journal of Cell Biology

Article Title: Lymphatic exosomes promote dendritic cell migration along guidance cues

doi: 10.1083/jcb.201612051

Figure Lengend Snippet: Primary human LECs release EEVs. (a) Transmitted EM (TEM) of human skin LECs in situ (left: antipodoplanin-conjugated gold particles) and in vitro (middle and right). MVBs (middle, unstained) and exocytosis of CD63 + vesicles (right, anti-CD63–conjugated gold particles) into the basolateral stroma ( n ≥ 5). (b) Experimental setup to collect basolateral LEC culture supernatant from the lower chamber for the enrichment of basolateral EEVs (yellow dots). (c) TEM of vesicles of basolateral EEV fractions. Right: Anti-CD63–conjugated gold particles ( n ≥ 21). Bars, 100 nm. (d) Diameters of vesicles in basolateral EEV fractions collected from ss ( n = 21) and TNFα-stimulated LECs ( n = 26). Data are obtained from at least seven TEM images per experiment and from three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). (e) BCA-derived protein concentrations of EEV fractions collected over 24 h from ss and TNFα-stimulated LECs in the absence or presence of 10 µM GW4869 ( n = 3; unpaired two-tailed t test). (f) Anti-CD9 immunoblot of EEV fractions from equal volumes of basolateral ss and TNFα-stimulated LEC culture supernatants ( n = 3). (g) Mean absolute vesicle numbers of EEV fractions isolated from 1 ml basolateral LEC culture supernatant of ss and TNFα-stimulated LECs ( n = 3; unpaired two-tailed t test). Left graph, vesicle numbers plotted against diameter size intervals; right graph, vesicle numbers of all sizes. (h) Coomassie blue–stained electrophoresis gel (top) and quantitation of relative protein abundance (as measured by densitometric analyses; bottom) of density gradient centrifugation subfractions of EEV fractions isolated from basolateral culture supernatants of ss and TNFα-stimulated LECs ( n = 3). (i) Anti-CD63–stained immunoblot (top) and quantitation of relative protein abundance (as measured by densitometric analyses; bottom) of density gradient centrifugation subfractions of EEV fractions isolated from basolateral culture supernatants of ss and TNFα-stimulated LECs ( n = 3). Values represent means ± SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01.

Article Snippet: Primary human dermal microvascular LECs (C12260; PromoCell) were expanded in endothelial cell growth medium-2 (EGM2MV; Lonza) until passage three, enriched for podoplanin + cells by flow cytometry, further expanded once and then stored in liquid N 2 .

Techniques: In Situ, In Vitro, Two Tailed Test, Derivative Assay, Western Blot, Isolation, Staining, Electrophoresis, Quantitation Assay, Quantitative Proteomics, Gradient Centrifugation

Journal: The Journal of Cell Biology

Article Title: Lymphatic exosomes promote dendritic cell migration along guidance cues

doi: 10.1083/jcb.201612051

Figure Lengend Snippet: Proteomic profiling reveals a migration-promoting protein signature in TNFα EEV fractions. (a–d) EEV fractions from basolateral culture supernatants of ss ( n = 3) or TNFα-stimulated LECs ( n = 3) were proteomically profiled with TMT-based LC-MSMS analysis. (a) Heat map of all significantly (P < 0.05) quantified proteins of EEV fractions. Three left lanes: Replicate EEV fractions from ss LECs; three right lanes: Replicate EEV fractions from TNFα-stimulated LECs. Values in heat maps are log 10 of interprotein abundance of a given sample divided by the mean abundance of the three ss samples. (b) Ratio density plot of EEV fractions from ss and TNFα-stimulated LECs. X axis: Log 10 ratio of interprotein abundance of TNFα-EEV fractions over ss-EEV fractions. Y axis: Relative number of proteins. (c) The quantitatively identified proteins of EEV fractions were grouped into biologically relevant clusters according to the databases of ExoCarta (exosomes), EVpedia (extracellular vesicles), and the Gene Ontology Consortium (cellular components). Mean interprotein abundances of proteins in the respective clusters were compared with the mean interprotein abundance of all proteins in ss-EEV fractions (red dotted line) and TNFα-EEV fractions (blue dotted line). X axis: Logarithmic scale of interprotein abundances. Y axis: Biologically relevant clusters. For each cluster, the floating bars display the minimum-to-maximum intervals of the interprotein abundance for ss-EEV (red) and TNFα-EEV (blue) fractions. Vertical lines show the mean interprotein abundance for ss-EEV (red) and TNFα-EEV (blue) fractions. **, P < 0.01; *** P ≤ 0.001. (d) Heat maps of significantly (P < 0.05) quantified proteins of EEV fractions that are either endothelial markers or chemokines and growth factors.

Article Snippet: Primary human dermal microvascular LECs (C12260; PromoCell) were expanded in endothelial cell growth medium-2 (EGM2MV; Lonza) until passage three, enriched for podoplanin + cells by flow cytometry, further expanded once and then stored in liquid N 2 .

Techniques: Migration

Journal: The Journal of Cell Biology

Article Title: Lymphatic exosomes promote dendritic cell migration along guidance cues

doi: 10.1083/jcb.201612051

Figure Lengend Snippet: Induction of protrusion formation and enhancement of directional migration by TNFα-EEV fractions is dependent on GPCR signaling and CX3CL1. (a) Quantitation of transmigrated human mature MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. PTX-treated or untreated MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions into the upper cell culture insert ( n = 3; unpaired two-tailed t test). (b) Immunofluorescence of CX3CL1 (green) and ALIX (red; left) or CD9 (red; right) in primary human LECs. Cell nuclei are stained with DAPI (blue; n = 5). Yellow regions of interest were used for Manders colocalization analyses. White region of interest is a zoom. Arrows indicate colocalization of CX3CL1 with either ALIX or CD9. (c) Immunofluorescence of the lymphatic vessel marker podoplanin (green) and CX3CL1 (red) in human renal transplant rejections (fluorescence profiles across the respective vessels are plotted beneath the merged images; x axis: cross-sectional distance; y axis: CX3CL1- or podoplanin-specific mean immunofluorescence intensities; n = 6). Bars, 5 µm. (d) Anti-CX3CL1 immunoblot of ss-EEV fractions and TNFα-EEV fractions probed with an antibody to the C terminus of CX3CL1 ( n = 5). Molecular masses are given in kilodaltons. (e) Flow cytometry contour plot of unstained beads, isotype control IgG-coated beads stained with fluorescent SYTO RNASelect-labeled TNFα-EEV fractions and anti-CX3CL1 IgG-coated beads stained with fluorescent SYTO RNASelect-labeled TNFα-EEV fractions. X axis indicates forward scatter. Y axis indicates mean fluorescence intensity (MFI) of SYTO RNASelect–labeled EEVs ( n = 6). (f) Quantitation of SYTO RNASelect MFI of beads described in e ( n = 6; unpaired two-tailed t test). (g) Quantitation of transmigrated MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions and control IgGs or anti-CX3CL1 IgGs into the upper cell culture insert ( n = 3; unpaired two-tailed t test). (h) Quantitation of transmigrated MMDCs from the upper cell culture insert into the lower chamber well of a transwell assay. MMDCs were loaded together with EEV-free supernatants or TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs or CX3CL1-specific morpholino oligonucleotide–treated LECs into the upper cell culture insert ( n = 2; unpaired two-tailed t test). (i) Migration analyses of MMDCs in a 3D collagen matrix migration assay. Cells were exposed to gradients of EEV-free supernatants plus CCL19 ( n = 1,070), TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs plus CCL19 ( n = 654) or TNFα-EEV fractions derived from CX3CL1-specific morpholino oligonucleotide–treated LECs plus CCL19 ( n = 958). Red columns indicate chemotactic displacement. Blue columns indicate chemotactic index. Data are obtained from at least 218 cell tracks per experiment and three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). (j) Migration analyses of MMDCs in a confined microenvironment migration assay in the presence of EEV-free supernatants ( n = 1,464), TNFα-EEV fractions derived from 5-mispair control morpholino oligonucleotide–treated LECs ( n = 9,380), or TNFα-EEV fractions derived from CX3CL1-specific morpholino oligonucleotide–treated LECs ( n = 5,588). Red columns indicate circularity of cell shape. Blue columns indicate migratory angle change. Green columns indicate speed of migration. Data are obtained from at least 488 time points and from three independent experiments that were pooled for analysis (unpaired two-tailed t test with Welch’s correction). Values represent means ± SEM. ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Primary human dermal microvascular LECs (C12260; PromoCell) were expanded in endothelial cell growth medium-2 (EGM2MV; Lonza) until passage three, enriched for podoplanin + cells by flow cytometry, further expanded once and then stored in liquid N 2 .

Techniques: Migration, Quantitation Assay, Cell Culture, Transwell Assay, Two Tailed Test, Immunofluorescence, Staining, Marker, Fluorescence, Western Blot, Flow Cytometry, Control, Labeling, Derivative Assay

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: ( A and B ) Transverse sections at the level of the anterior CV show that expression of Vegfr3 is severely reduced in differentiating Prox1-expressing LECs outside the CV. Similarly, other LEC markers such as Pdpn ( C and D ) and Lyve1 ( E and F ) are also down-regulated, whereas expression of the pan-endothelial markers VEcad ( G and H ) and CD31 ( I and J ) is normal. White box insets correspond to higher magnification of the dotted box region in each panel. Arrows indicate dorsal (D) and lateral (L) orientations. Mean fluorescence intensity of each staining was quantified and shown as percentage of mean of control group in ( K ) and ( L ). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by multiple t test. Scale bar, 50 μm ( n = 6).

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Expressing, Fluorescence, Staining, Control

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: ( A to J ) At E12.5, expression of Prox1 still remains in the mutant embryos; however, expression of most other LEC markers is barely detected or is severely down-regulated. Expression of the pan-endothelial marker CD31seems normal. White arrows in control embryo (E) point to the dermal lymphatic vasculature; mutant LECs fail to sprout from the lymph sacs such that lymph sacs (LS) become abnormally enlarged and mutant embryos lack dermal lymphatics. White box insets correspond to higher magnifications of the regions inside the dotted boxes in each panel. Mean fluorescence intensity of each staining was quantified and shown as percentage of mean of control group in ( K ). Each data point represents a biological replicate. Area of the CV and lymph sac was quantified and shown as fold change of lymph sac area compared to CV in each genotype ( L ). All results are presented as means ± SEM and analyzed by multiple t test for (K) and two-tailed Student’s t test for (L). Scale bar, 50 μm ( n = 3 to 8).

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Expressing, Mutagenesis, Marker, Control, Fluorescence, Staining, Two Tailed Test

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: ( A and B ) At E11.5, QPC f/f ;PdpnGFPCre LECs show severely reduced Vegfr3 levels, but similar to controls, Prox1 + LECs do not coexpress BEC markers such as CD34 ( n = 3). ( C and D ) At around E12.5, Vegfr3 expression becomes undetectable in mutant LECs and Prox1 + (low) LECs abnormally up-regulate the expression of CD34, an indication that they are losing LEC fate and regaining BEC fate. White arrows indicate Prox1 + CD34 + LECs. Blood cells are abnormally seen inside the mutant lymph sacs (D), most likely a consequence of defective lymphovenous valves. White box insets correspond to higher magnification of the regions in dotted boxes in each panel. CD34 + Prox1 + cells were quantified and shown as percentage of Prox1 + in each group ( E ). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by two-tailed Student’s t test. Scale bar, 50 μm ( n = 6).

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Expressing, Mutagenesis, Two Tailed Test

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: ( A ) Antimycin A (Anti) treatment reduces Prox1 ( n = 9), Vegfr3 ( n = 9), and Nrp2 ( n = 5) mRNA levels but not those of VEcad ( n = 3). ( B ) Myxothiazol (Myx) treatment also reduces Prox1 ( n = 4), Vegfr3 ( n = 6), and Nrp2 ( n = 3) mRNA levels but not those of VEcad ( n = 3). ( C ) Representative Western blot shows that antimycin A and myxothiazol treatment reduces VEGFR3 ( n = 6) and PROX1 ( n = 6) levels but not those of VECAD ( n = 5 to 6). ( D ) Quantification of the densitometry of each protein normalized to GAPDH and shown as fold change compared to controls. Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by multiple t test in (A) and (B) or two-way analysis of variance (ANOVA) in (D). ( E and F ) RNA-seq of LECs treated with vehicle or antimycin A shows that expression of most LEC genes in the dataset is reduced, while almost half of the BEC genes are up-regulated, including Nrp1 and ICAM1 ( n = 3).

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Western Blot, RNA Sequencing, Expressing

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: Antimycin A inhibits OCR ( A ), NAD + /NADH ratio ( B ), and cell proliferation ( C ) in LECs transduced with EV-GFP, while AOX rescues OCR ( n = 3 to 4) (A), NAD + /NADH ratio ( n = 3) (B), and cell proliferation ( n = 3) (C) in the presence of antimycin A. AOX also rescued the Vegfr3 , Prox1 , and Nrp2 levels in the presence of antimycin A ( D ) ( n = 5 to 6). Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by one-way ANOVA for (A) to (C) or two-way ANOVA for (D).

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Transduction

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: Mitochondrial complex III inhibition with antimycin A alters TCA cycle metabolites ( A ) and 2HG levels ( B ). Values are normalized to mean of control group. ( C ) ChIP-seq analysis of H3K27ac, H3K4me3, and H3K4me1 histone modifications in LEC cultures treated with vehicle control (Ctrl) or antimycin A (Anti) for 48 hours. Track examples for LEC-specific genes ( Prox1 and Vegfr3 ) and BEC genes ( Nrp1 and ICAM1 ) are shown. Antimycin A–treated LECs show a marked reduction in the H3K4me3 and H3K27ac signal at the Vegfr3 locus and a reduction in H3K4me3 in the Prox1 locus, while H3K27ac peaks are increased in the Nrp1 and ICAM1 locus. ( D ) ChIP-seq analysis of the same modifications in (C) in LECs and BECs reveals that H3K27ac and H3K4me3 peaks are elevated in the Prox1 and Vegfr3 loci in LECs, whereas those of Nrp1 and ICAM1 are reduced in LECs and BECs. Scale bar, 5 kb. Each data point represents a biological replicate. All results are presented as means ± SEM and analyzed by multiple t test or two-tailed Student’s t test.

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Inhibition, Control, ChIP-sequencing, Two Tailed Test

Journal: Science Advances

Article Title: Mitochondrial respiration controls the Prox1-Vegfr3 feedback loop during lymphatic endothelial cell fate specification and maintenance

doi: 10.1126/sciadv.abe7359

Figure Lengend Snippet: Venous ECs (VECs) inside the CV are glycolytic. Starting at around E9.5, CoupTFII and Sox18 induce Prox1 expression in some VECs inside the CV to give rise to Prox1 + LEC progenitors. Specification of LEC fate, LEC budding, and maintenance of LEC fate is regulated by a Prox1-Vegfr3 feedback loop. Prox1 also regulates CPT1a, which, in turn, increases FAO in LECs. FAO-derived acetyl-CoA promotes histone acetylation by the histone acetyltransferase P300 and Prox1 complex at lymphangiogenic genes such as Vegfr3 . In addition, ketone body oxidation mediated by 3-oxoacid-CoA-transferase-1 (OXCT1) also generates acetyl-CoA, regulates TCA cycle metabolites and aspartate and dNTP levels, and is required for LEC proliferation. We demonstrate that mitochondrial respiratory chain is required for LEC fate maintenance. When complex III is blocked, mutant embryos lack LECs as a consequence of loss of Vegfr3 expression and LEC fate. Mitochondrial respiration is required for nucleotide synthesis and the maintenance of H3K4me3 and H3K27ac histone modifications at the Vegfr3 and Prox1 promoters. Blockage of mitochondrial respiration reduced Vegfr3 and disrupted the Vegfr3-Prox1 feedback loop.

Article Snippet: Primary human dermal microvascular LECs and HUVECs were purchased from Lonza.

Techniques: Expressing, Derivative Assay, Mutagenesis