Journal: bioRxiv

Article Title: AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

doi: 10.1101/2020.10.16.342998

Figure Lengend Snippet: ( A and B ). Cholesterol removal potentiates VEGFR3 signaling. ( A ) hLECs were serum-starved, and treated with 10 mM MβCD for 30 min, and cells were further stimulated with 100 ng/ml VEGFC. The resulting cells were lysed and blotted with Cav-1 or GAPDH antibodies. ( B ) hLECs were treated as in ( A ). and cell lysates were immunoprecipitated using VEGFR3 antibody. Immunoblotting was performed using anti-phosphotyrosine (4G10) and VEGFR3 antibodies. ( C ) AIBP-mediated cholesterol efflux disrupts caveolae and reduces CAV-1 levels in the caveolar fractions. hLECs were treated with recombinant 200 ng/ml AIBP, 100 μg/ml HDL 3 , or both in serum-free EBM2 for 6 hours, and the cells were subjected to sucrose-mediated ultracentrifugation. The resulting fractions were collected for Western blot analysis as indicated. ( D ) AIBP-mediated cholesterol efflux increases VEGFR3 signaling. hLECs were serum-starved and treated as in ( C ), and further stimulated with 100 ng/ml VEGFC. The resulting cells were lyzed and immunoblotted as indicated. ( E and F ), Quantitative data of AKT activation ( E ) and ERK activation ( F ). Mean±SD, n=3 independent repeats. *, p<0.05; **, p<0.01; ns: not significant.

Article Snippet: Human dermal lymphatic microvascular endothelial cells (hLECs) (Angio-Proteomie, Cat # cAP-0003) were cultured in Endothelial Cell Medium (ScienCell, Cat # 1001).

Techniques: Immunoprecipitation, Western Blot, Recombinant, Activation Assay

Journal: bioRxiv

Article Title: AIBP-CAV1-VEGFR3 axis dictates lymphatic cell fate and controls lymphangiogenesis

doi: 10.1101/2020.10.16.342998

Figure Lengend Snippet: ( A ) Conserved CAV-1 binding site on VEGFR3 in human (Hu), mouse (Ms), and zebrafish (Zf). ( B ) VEGFR3 AAA loses its binding to CAV-1. hLECs were transfected with control EGFP (Ctrl), VEGFR3-EGFP (R3), or VEGFR3 AAA -EGFP (R3 AAA ) using lentivirus-mediated gene transfer. After 72 hours, the resulting cells were lyzed and immunoprecipitated with EGFP antibody coupled to the magnetic Dynabeads and immunoblotted using VEGFR3 and CAV-1 antibodies. Immunoblotting of overexpressed VEGFR3-EGFP and VEGFR3 AAA -EGFP (R3/R3 AAA -EGFP) were detected using VEGFR3 antibody. The input lysates were shown on the right. ( C ) VEGFR3 AAA increases VEGFR3 signaling. hLECs were transduced as in ( B ), and the resulting cells were serum starved and treated with 100 ng/ml VEGFC for 20 min, cells were then lysed and immunoblotted as indicated. Quantitative data of VEGFR3 activation ( D ), AKT activation ( E ), and ERK activation ( F ) were shown. n=3 independent repeats. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: Human dermal lymphatic microvascular endothelial cells (hLECs) (Angio-Proteomie, Cat # cAP-0003) were cultured in Endothelial Cell Medium (ScienCell, Cat # 1001).

Techniques: Binding Assay, Transfection, Control, Immunoprecipitation, Western Blot, Activation Assay

Journal: Pharmaceuticals

Article Title: Use of Early Donated COVID-19 Convalescent Plasma Is Optimal to Preserve the Integrity of Lymphatic Endothelial Cells

doi: 10.3390/ph15030365

Figure Lengend Snippet: Elevated antibody concentrations and prolonged symptoms are detrimental for the lymphatic endothelium integrity. ( A ) Correlation between the duration of symptoms (square-root transformation) and the concentration of plasmatic SARS-CoV-2- receptor-binding-domain antibodies measured by ELISA. ( B ) Correlation between the duration of symptoms and MitoSOX TM Red-negative cells measured by flow cytometry after incubating aHDLEC with convalescent plasma for 4 h and cytokines cocktail for 20 h. ( C ) Correlation between the concentration of plasmatic RBD antibodies measured by ELISA and the permeability of the endothelium measured by spectrophotometry (absorbance of OVA-488) after incubating human LEC with convalescent plasma for 4 h and cytokines cocktail for 20 h. Each point represents a treatment. Significance ( p < 0.05) was determined by a Pearson correlation. A square-root transformation of the duration of symptoms to reach normal distribution was performed. RBD—receptor-binding domain; O.D—optical density; OVA—ovalbumin; aHDLEC—adult human dermal lymphatic endothelial cells.

Article Snippet: Primary adult human dermal lymphatic endothelial cells (aHDLEC, PromoCell, cat. C-12217) were used in this study and called adult human lymphatic endothelial cells (aHDLEC) throughout the manuscript.

Techniques: Transformation Assay, Concentration Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Permeability, Spectrophotometry

Journal: Pharmaceuticals

Article Title: Use of Early Donated COVID-19 Convalescent Plasma Is Optimal to Preserve the Integrity of Lymphatic Endothelial Cells

doi: 10.3390/ph15030365

Figure Lengend Snippet: The severity of symptoms correlates with the duration of symptoms, age, receptor-binding-domain antibodies of donors and cell viability of lymphatic endothelial cells. ( A ) Severity was quantified using a questionnaire answered by donors and graded as follows: asymptomatic, mild, moderate and severe. Severe symptoms were correlated with a higher duration of symptoms (square-root transformation to reach a normal distribution). ( B ) Correlations between severity of the symptoms and the age of the donors were performed. ( C ) Severity was correlated with the concentration of antibodies measured by ELISA. ( D , E ) Treated aHDLEC were labeled with Annexin V and PI and analyzed by flow cytometry. The severity of the symptoms was correlated with cells in late apoptosis/necrosis ( D ) and in early apoptosis ( E ) represented by Annexin V- and PI-positive cells and Annexin V-positive and PI-negative cells, respectively. Cells are given as percentages relative to cells treated with control plasma. Significance was determined by a Spearman correlation. p < 0.05 was considered significant. RBD, receptor-binding domain; O.D, optical density; PI, propidium iodide; aHDLEC, adult human dermal lymphatic endothelial cells.

Article Snippet: Primary adult human dermal lymphatic endothelial cells (aHDLEC, PromoCell, cat. C-12217) were used in this study and called adult human lymphatic endothelial cells (aHDLEC) throughout the manuscript.

Techniques: Binding Assay, Transformation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Labeling, Flow Cytometry

Journal: Pharmaceuticals

Article Title: Use of Early Donated COVID-19 Convalescent Plasma Is Optimal to Preserve the Integrity of Lymphatic Endothelial Cells

doi: 10.3390/ph15030365

Figure Lengend Snippet: Early donation of COVID-19 convalescent plasma is a good predictor of preserved endothelial integrity. ( A , B ) Treated aHDLEC (convalescent plasma for 4 h and cytokines for 20 h) were harvested and the mRNA expression, measured by RT-qPCR, of SELE ( A ) and ICAM1 ( B ) was correlated with the time of donation since the onset of symptoms. ( C ) Following treatment, cells were fixed in paraformaldehyde (PFA) 2% and incubated with anti-VE-Cadherin antibodies. The intensity of VE-Cadherin relative to cells treated with control plasma was correlated to the duration between the onset of symptoms and the donation. ( D ) Immunofluorescence images of treated aHDLEC incubated with CCP donated at 27 days (upper panels) and 101 days (lower panels) post onset of symptoms. The left panels show the expression of VE-Cadherin and DAPI, whereas the middle panel represents the WGA staining. The right panels show the representation of the merged staining. Scale bar = 50 μm. ( E ) VE-Cadherin mRNA expression was correlated to the duration between the onset of symptoms and donation (∆). ( F ) Endothelial permeability was analyzed by the relative concentration of ovalbumin-488 measured following migration through the endothelium compared to control. The concentration of OVA-488 was correlated with the duration between the onset of symptoms and the donation. Significance was determined by Pearson correlation and p < 0.05 was considered significant. ∆, time of donation since onset of symptoms; SELE , gene coding for E-selectin; ICAM1 , gene coding for intercellular adhesion molecule 1; WGA, wheat germ agglutinin; CDH5 , gene coding for VE-Cadherin; OVA, ovalbumin; aHDLEC, adult human dermal lymphatic endothelial cells; CCP, COVID-19 convalescent plasma.

Article Snippet: Primary adult human dermal lymphatic endothelial cells (aHDLEC, PromoCell, cat. C-12217) were used in this study and called adult human lymphatic endothelial cells (aHDLEC) throughout the manuscript.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Immunofluorescence, Staining, Permeability, Concentration Assay, Migration

Journal: Pharmaceuticals

Article Title: Use of Early Donated COVID-19 Convalescent Plasma Is Optimal to Preserve the Integrity of Lymphatic Endothelial Cells

doi: 10.3390/ph15030365

Figure Lengend Snippet: Secretion of lymphatic endothelial cells derived extracellular vesicles is a marker of impaired integrity. ( A ) Treated aHDLEC (convalescent plasma for 4 h and cytokines for 20 h) labeled by the MitoSOX TM Red probe and correlated with the quantity of EVs shed by the lymphatic endothelial cells. ( B , C ) Cells in sub-G 0 /G 1 ( B ) and G 2 /M ( C ) phases of the cell cycle, determined by cell cycle analysis using PI, were correlated to the quantity of EVs secreted by the lymphatic endothelium. ( D , E ). Treated aHDLEC were harvested and expression of FLT4 was assessed by RT-qPCR ( D ) and immunoblot ( E ) followed by a normalization onto the expression of ACTB or α/ß tubulin, respectively. The measured expression of VEGFR-3 was then correlated with the quantity of EVs secreted by the endothelium. ( F ) Immunoblot analysis for VEGFR-3 and the loading control α/ß tubulin. The left panel represents aHDLEC secreting low quantities of CD45 − podoplanin + EVs (less than 120,000 EVs) following incubation with CCP, and the right represents aHDLEC secreting high quantities of CD45 − podoplanin + EVs (more than 380,000 EVs). Significance was determined by a Pearson correlation and p < 0.05 was considered significant. EVs, extracellular vesicles; PI, propidium iodide; CCP, COVID-19 convalescent plasma; VEGFR-3, vascular endothelial growth factor receptor 3; FLT4, gene coding for VEGFR-3; aHDLEC, adult human dermal lymphatic endothelial cells; AU, arbitrary unit.

Article Snippet: Primary adult human dermal lymphatic endothelial cells (aHDLEC, PromoCell, cat. C-12217) were used in this study and called adult human lymphatic endothelial cells (aHDLEC) throughout the manuscript.

Techniques: Derivative Assay, Marker, Labeling, Cell Cycle Assay, Expressing, Quantitative RT-PCR, Western Blot, Incubation