Journal: PLoS ONE

Article Title: Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

doi: 10.1371/journal.pone.0136863

Figure Lengend Snippet: Responses of leukocyte rolling velocity (A), rolling flux (B) and adhesion (C) to endothelial monolayer were monitored using 40x objective lens of an inverted microscope connected to a video camera. Data presented are mean ± SEM, n≥4, *p<0.05, **p<0.01 vs. corresponding shc002 cells. (D) Whole cell lysates were immunoblotted with antibodies against VCAM-1, ICAM-1 and VDR. The same samples were reprobed with tubulin to ensure equal loading. Representative Western blot (D) and quantitative analysis (E) demonstrate decrease in VDR expression in EA.hy926 infected with pLKO.1-VDR (shVDR) compared with pLKO.1-SHC002 (shc002 control). Data presented are mean ± SEM from 3 independent experiments, ***p<0.005 vs. shc002. Downregulation of VDR in endothelial cells resulted in increased VCAM-1 (D, F) and ICAM-1 (D, G) levels compared to control cells. *p<0.05 vs. control.

Article Snippet: Briefly, real time PCR with gene-specific TaqMan probes for mouse VCAM-1 (Mm01320970_m1), ICAM-1 (Mm00516023_m1), IL-6 (Mm00446190_m1) (Applied Biosystems-Life Technologies S.A., Madrid, Spain) and human IL-6 (Hs.PT.58.40226675) (Integrated DNA Tech, Inc., Madrid, Spain) was performed with a CFX Real-Time PCR detection system (Bio-Rad Laboratories, S.A., Madrid, Spain) using TaqMan Universal PCR Master Mix, No AmpErase UNG.

Techniques: Inverted Microscopy, Western Blot, Expressing, Infection, Control

Journal: PLoS ONE

Article Title: Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

doi: 10.1371/journal.pone.0136863

Figure Lengend Snippet: EA.hy926 cells (shc002 or shVDR) were untreated (A, B, C) or treated with IκB kinase (IKK) inhibitor PS-1145 (D) for 24 hours. (A, B, D) Whole cell lysates were immunoblotted with antibodies against VDR, IκB-α, VCAM-1 and ICAM-1. The same samples were reprobed with tubulin to ensure equal loading. (C) Western blot analysis of p65 levels in cytosolic and nuclear extracts isolated from shc002 and shVDR endothelial cells. The nuclear protein Histone 1, which is absent in the cytosolic fraction, served as a nuclear protein loading control. (D) Representative Western blot and quantitative analysis (G, H), *p<0.05 vs. shc002 or shVDR, respectivey (E) Real time PCR analysis of IL-6 mRNA in shc002 and shVDR endothelial cells. Data presented are mean ± SEM from 3 independent experiments. **p<0.01 vs. shc002. (F) Determination of IL-6 secretion into the medium by ELISA. Endothelial cells were grown in normal growth media for 72 hours. IL-6 production was determined using a human IL-6 HS ELISA kit. Data presented are mean ± SEM from 3 independent experiments. *p<0.05 vs. shc002.

Article Snippet: Briefly, real time PCR with gene-specific TaqMan probes for mouse VCAM-1 (Mm01320970_m1), ICAM-1 (Mm00516023_m1), IL-6 (Mm00446190_m1) (Applied Biosystems-Life Technologies S.A., Madrid, Spain) and human IL-6 (Hs.PT.58.40226675) (Integrated DNA Tech, Inc., Madrid, Spain) was performed with a CFX Real-Time PCR detection system (Bio-Rad Laboratories, S.A., Madrid, Spain) using TaqMan Universal PCR Master Mix, No AmpErase UNG.

Techniques: Western Blot, Isolation, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

doi: 10.1371/journal.pone.0136863

Figure Lengend Snippet: Responses of leukocyte rolling velocity (A), rolling flux (B) and adhesion (C) to endothelial monolayer were monitored using 40x objective lens of an inverted microscope connected to a video camera. Data presented are mean ± SEM, n≥4, **p<0.01, ***p<0.005, *#p<0.001. (D) Whole cell lysates were immunoblotted with antibodies against VCAM-1, ICAM-1, IκBα and VDR. The same samples were reprobed with tubulin to ensure equal loading. Representative Western blot (D) and quantitative analysis (E, F) *p<0.05 (VCAM-1) or **p<0.01 (ICAM-1) vs. shc002/Control; **p<0.01 (VCAM-1) or *#p<0.001 (ICAM-1) vs. shVDR/Control. (G) Real time PCR analysis of IL-6 mRNA. Data presented are mean ± SEM from 3 independent experiments. *p<0.05. (H) Determination of IL-6 secretion into the medium by ELISA. Endothelial cells were grown in normal growth media for 72 hours. IL-6 production was determined using a human IL-6 HS ELISA kit. Data presented are mean ± SEM from 3 independent experiments. *p<0.05, *#p<0.001, **#p<0.0001.

Article Snippet: Briefly, real time PCR with gene-specific TaqMan probes for mouse VCAM-1 (Mm01320970_m1), ICAM-1 (Mm00516023_m1), IL-6 (Mm00446190_m1) (Applied Biosystems-Life Technologies S.A., Madrid, Spain) and human IL-6 (Hs.PT.58.40226675) (Integrated DNA Tech, Inc., Madrid, Spain) was performed with a CFX Real-Time PCR detection system (Bio-Rad Laboratories, S.A., Madrid, Spain) using TaqMan Universal PCR Master Mix, No AmpErase UNG.

Techniques: Inverted Microscopy, Western Blot, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

doi: 10.1371/journal.pone.0136863

Figure Lengend Snippet: (A) Effects of VDR deletion on proinflammatory cytokine production. Circulating serum levels of interleukin (IL-6) were determined by ELISA. Data presented are mean ± SEM of n = 10 mice/group. *p<0.05 vs. apoE -/- . (B, C, D, E, F) Real time PCR analysis of VCAM-1, ICAM-1 and IL-6 mRNA in the aortic tissue of apoE -/- and apoE -/- VDR -/- mice (B, C, D) and VDR +/+ and VDR -/- mice (E, F). Data presented are mean ± SEM. (B, C) *p<0.05 vs. apoE -/- ; (E, F) *p<0.05 vs. VDR +/+ mice. (G) Enhanced VCAM-1 and ICAM-1 expression in the vascular endothelium of apoE -/- VDR -/- mice. Aortic root lesions from apoE -/- and apoE -/- VDR -/- mice were stained with anti-VCAM-1 and anti-ICAM-1. CD31 was used for endothelial cell staining. Representative images are shown in G. Magnification 20x.

Article Snippet: Briefly, real time PCR with gene-specific TaqMan probes for mouse VCAM-1 (Mm01320970_m1), ICAM-1 (Mm00516023_m1), IL-6 (Mm00446190_m1) (Applied Biosystems-Life Technologies S.A., Madrid, Spain) and human IL-6 (Hs.PT.58.40226675) (Integrated DNA Tech, Inc., Madrid, Spain) was performed with a CFX Real-Time PCR detection system (Bio-Rad Laboratories, S.A., Madrid, Spain) using TaqMan Universal PCR Master Mix, No AmpErase UNG.

Techniques: Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing, Staining

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a-c) Adoptive transfer experiments; a 1:1 mixture of PKH26-red labeled Cre - α 4 f/f and PKH67-green labeled Cre + α 4 f/f cells (monocytes or splenic T cells) was simultaneously transferred into obese wild-type mice. The numbers of Cre - α 4 f/f and Cre + α 4 f/f macrophages ( a ) or T cells ( c ) accumulated in inguinal subcutaneous adipose tissue (SAT) or visceral adipose tissue (VAT) and the numbers of Cre - α 4 f/f and Cre + α 4 f/f monocytes/macrophages accumulated in the lymph nodes draining the SAT (SAT dLN) and lymph nodes draining the VAT (VAT dLN) ( b ) were analyzed by flow cytometry. Data (percentage of labeled cells in defined cells) are presented as relative to control. The percentage of labeled Cre - α 4 f/f cells in defined cells was set as 100% in each tissue; n=5 recipient mice in a,b and n=4 recipient mice in c ; data in ( b ) are from one experiment, data in ( a ), ( c ) are representative of two experiments. d) Vcam1 mRNA expression was determined in VAT and SAT of lean mice, fed a normal diet (ND, n=8 mice) or obese mice (fed a HFD, n=10 mice), in mature adipocytes from VAT and SAT of lean (ND, n=6 mice) or obese mice (HFD, n=6 mice), and in CD31 + endothelial cells from SAT of lean (ND, n=5 mice) or obese mice (HFD, n=6 mice). 18S expression was used for normalization and Vcam1 expression of ND-fed mice was set as 1 in each case. Data are from one experiment (SAT endothelial cells) or representative of two experiments (VAT, SAT, VAT adipocytes, SAT adipocytes). ( e ) Comparison of Vcam1 mRNA expression between CD45 - CD31 + endothelial cells and adipocytes from SAT of obese mice is shown. Vcam1 expression of endothelial cells was set as 1; n=6 mice; data are from one experiment. f) Primary mouse adipocytes were treated without (control; Con) or with TNF or palmitate. The surface expression of VCAM-1, expressed as Median Fluorescence Intensity (MFI) units, analyzed by flow cytometry, is shown (n=6 separate cell isolations; data are representative of two experiments). In the right panel, a representative flow cytometry plot depicting VCAM-1 expression of Control-, TNF-, or palmitate-treated adipocytes, as well as isotype control (Iso), is shown. g) Adhesion assay of bone marrow mononuclear cells (BMM) to TNF- or palmitate-pretreated 3T3-L1 adipocytes was performed in the presence of ICAM-1- or VCAM-1-blocking antibodies or respective isotype controls (ICAM-1 Iso or VCAM-1 Iso). The percentage of adherent cells is shown. Shown is one experiment performed in triplicate; representative of 3 experiments. h) Adhesion of BMM from Cre + α 4 f/f or Cre - α 4 f/f mice to TNF- or palmitate-pretreated 3T3-L1 adipocytes (data from separate BMM isolations from n=6 Cre + α 4 f/f mice and n=8 Cre - α 4 f/f mice; pooled from 3 experiments). The percentage of adherent cells is shown. Data are presented as mean ± SEM. *P < 0.05. Mann-Whitney U-test in ( a ), ( c ), ( d ), ( e ), ( f ), Student's t-test in ( b ), ( g ), ( h ).

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Adoptive Transfer Assay, Labeling, Flow Cytometry, Control, Expressing, Comparison, Fluorescence, Cell Adhesion Assay, Blocking Assay, MANN-WHITNEY

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a) Energy expenditure (EE, expressed as kcal/h) of obese Cre + α 4 f/f (n=4 mice) and Cre - α 4 f/f (n=5 mice) mice was assessed by a metabolic cage monitoring system for 3 days (left). Average EE in light and dark period is also displayed (right). b) Gene expression in SAT of obese Cre + α 4 f/f or Cre - α 4 f/f mice was evaluated by qPCR (n=8 mice for Cre + α 4 f/f and 10-11 mice for Cre - α 4 f/f ). 18S expression was used for normalization of mRNA expression and the respective gene expression of obese Cre - α 4 f/f mice was set as 1. c) Representative cropped blot images showing immunoblotting for UCP1 (and actin) in SAT from 2 obese Cre + α 4 f/f and 2 Cre - α 4 f/f mice. Actin was used as loading control. d-f) Obese Cre + α 4 f/f or Cre - α 4 f/f mice were challenged with a temperature of 4°C for 12 h. d) Core temperature of obese Cre + α 4 f/f (n=4 mice) or Cre - α 4 f/f (n=6 mice) mice during cold exposure. e) Representative cropped blot images showing immunoblotting (left) for UCP1 (and actin) in SAT of 2 obese Cre + α 4 f/f and 2 obese Cre - α 4 f/f mice exposed to cold. Densitometric analysis (right) of UCP1 immunobloting from a total of 4 Cre + α 4 f/f mice and 5 Cre - α 4 f/f mice is shown. The protein amounts of UCP1 were normalized against actin; the UCP1 amounts (normalized over actin) in SAT from Cre - α 4 f/f mice were set as 1. f) UCP1 staining in SAT from Cre + α 4 f/f or Cre - α 4 f/f mice exposed to cold. g) Mature white and brown adipocytes were isolated from SAT and BAT of obese mice (n=6 mice) after tissue digestion and Vcam1 mRNA expression was determined. 18S expression was used for normalization and Vcam1 expression of SAT was set as 1. Data are presented as mean ± SEM. Data in ( a ), ( d ) and ( e ) are from one experiment; data in ( b ) are pooled from 3 experiments; data in ( c ) and ( g ) are representative of 2 experiments. In ( f ) representative images are from analysis performed on 5 Cre - α 4 f/f and 4 Cre + α 4 f/f mice. *P < 0.05. ANCOVA in ( a ) and Mann-Whitney U-test in ( b ) – ( g ). Abbreviations: Ucp1 , uncoupling protein 1; Cidea , cell death-inducing DNA fragmentation factor-like effector A; Cox8b , cytochrome c oxidase subunit 8b; Cox7a1 , Cytochrome c oxidase subunit 7A1; Acsl1 , acyl-CoA synthetase long-chain family member 1

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Gene Expression, Expressing, Western Blot, Control, Staining, Isolation, MANN-WHITNEY

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a-b) Primary adipocytes were cultured with T3 and norepinephrine to induce a thermogenic response, prior to co-culture in the absence or presence of ( a ) M1-like polarized pro-inflammatory BMDMs or ( b ) M1-like polarized primary SAT-derived macrophages [M1MΦ in the figure are M1-like polarized BMDMs in ( a ) or SAT macrophages in ( b )] for 1 h. Co-culture was performed in direct contact (in the same well). Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). a ) Ucp1 mRNA expression was analyzed in adipocytes after separation from the BMDMs. 18S expression was used for normalization and Ucp1 expression in adipocytes cultured in the absence of macrophages and in the absence of α 4 -inhibitor was set as 1 (n=5 separate primary adipocyte isolations). b ) The mRNA expression of Ucp1 was analyzed in adipocytes after separation from SAT macrophages. 18S expression was used for normalization and Ucp1 expression in adipocytes cultured in the absence of macrophages and in the absence of α 4 -inhibitor was set as 1 (n=5 separate primary cell isolations from SAT). c-d) Primary adipocytes (pre-stimulated with T3 and norepinephrine) were co-cultured in the absence or presence of M2-like polarized primary SAT-derived macrophages (M2MΦ) in direct contact ( c , in the same well) or in an indirect way ( d , in transwell co-culture system, wherein adipocytes and macrophages were in the lower and upper compartment, respectively). Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The mRNA expression of Ucp1 was analyzed in adipocytes after separation from the macrophages. 18S expression was used for normalization and Ucp1 expression in adipocytes cultured in the absence of macrophages was set as 1 (n=5 separate primary cell isolations from SAT). e-f) Primary adipocytes (pre-stimulated with T3 and norepinephrine) were co-cultured in the absence (control; Con) or presence of M1-like polarized BMDMs (direct contact) for 30 min. The amounts of phospho-p38 ( e ) and phospho-Erk ( f ) in adipocytes were detected by flow cytometry. Phospho-p38 or phospho-Erk protein amounts (MFI) in adipocytes cultured in the absence of M1MΦ were set as 1 (n=4 separate primary adipocyte isolations). g) Primary adipocytes were pre-treated with T3 and norepinephrine for 3 h, washed and then incubated in the absence (control; Con) or presence of an Erk inhibitor for 30 min. After washing, direct co-cultures between adipocytes and M1-like BMDMs were performed for 1 h and the mRNA expression of Ucp1 was studied in adipocytes after separation from the macrophages. The mRNA expression of Ucp1 in the absence of BMDMs and without Erk-inhibitor pre-treatment was set as 1 (n=4 separate primary adipocyte isolations). h, i) T3- and norepinephrine-pre-treated adipocytes were co-cultured with M1-like BMDMs (M1MΦ) in direct contact for 30 min in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). Phospho-p38 ( h ) and phospho-Erk ( i ) in adipocytes were detected by flow cytometry and their respective amount (MFI) in the presence of M1MΦ but in the absence of α 4 -inhibitor was set as 1 (n=4 separate primary adipocyte isolations). j) Primary adipocyte progenitor cells (AP) were isolated from SAT of wild-type C57BL/6 lean (ND, n=4 mice) and obese (HFD, n=6 mice) mice by FACS-sorting (AP cells were defined as CD31 - CD45 - Sca1 + PDGFRα + cells) and Vcam1 mRNA expression was determined. 18S expression was used for normalization and Vcam1 expression of ND-fed mice was set as 1. k) AP were isolated from SVF of SAT of wild-type mice with FACS-sorting (CD31 - CD45 - Sca1 + PDGFRα + cells). Cells were treated without (control; Con) or with TNF. Vcam1 mRNA expression was analyzed. 18S expression was used for normalization and Vcam1 expression of control-treated cells (Con) was set as 1 (n=4 separate cell isolations). l) AP were isolated as in ( k ) and treated with T3 and norepinephrine. Thereafter, AP were co-cultured in the absence or presence of M1-like polarized pro-inflammatory BMDMs (M1MΦ) in direct contact with each other. Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The mRNA expression of Ucp1 in AP was detected after macrophage separation. 18S expression was used for normalization and Ucp1 expression in the absence of macrophages and in the absence of α 4 -inh. was set as 1 (n=5 separate primary adipocyte progenitor isolations). Data are presented as mean ± SEM. Data in ( b ), ( c ), ( d ) are pooled from 3 experiments; data in ( a ), ( e ), ( f ), ( g ), ( h ), ( i ), ( k ) are representative of 2 experiments; data in ( j ) and ( l ) are from one experiment. *P < 0.05. Mann-Whitney U-test in ( a )-( l ).

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Cell Culture, Co-Culture Assay, Derivative Assay, Control, Expressing, Flow Cytometry, Incubation, Isolation, MANN-WHITNEY

Journal: Nature immunology

Article Title: A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity

doi: 10.1038/ni.3728

Figure Lengend Snippet: a-d ) Peritoneal macrophages were FACS-sorted from wild-type C57BL/6 mice or Cre + α 4 f/f and Cre - α 4 f/f mice. a-b) Macrophages were cultured on plates pre-coated with Fc or VCAM-1-Fc. a) Wild-type macrophages (n=4 separate cell isolations) were incubated on pre-coated plates for 12 h in the absence (control; Con) or presence of the α 4 -inhibitor (α 4 -inh.) and macrophage TNF expression was analyzed by flow cytometry. Data are expressed as percentage of TNF-positive macrophages (F4/80 + CD11b + ). b) Isolated Cre + α 4 f/f (n=4 separate cell isolations) or Cre - α 4 f/f (n=5 separate cell isolations) macrophages were incubated on plates pre-coated with Fc or VCAM-1-Fc for 6 h and TNF expression was analyzed by flow cytometry. Data are presented as percentage of TNF-positive macrophages (F4/80 + CD11b + ). c-d) Macrophages were co-cultured in a direct cell-to-cell contact mode with 3T3-L1 adipocytes for 12 h. c) Wild-type macrophages were co-cultured with adipocytes in the absence (control, Con) or presence of α 4 -inhibitor (α 4 -inh.) and macrophage TNF expression was analyzed by flow cytometry. Data are presented as percentage of TNF-positive macrophages (defined as CD45 + F4/80 + CD11b + ) (n=4 separate macrophage isolations). d) Cre + α 4 f/f or Cre - α 4 f/f macrophages were co-cultured with adipocytes in a direct cell-to-cell contact mode and macrophage TNF expression was analyzed by flow cytometry. Data are expressed as percentage of TNF-positive macrophages (CD45 + F4/80 + CD11b + ) (n=4 separate macrophage isolations). Data in ( a )-( d ) are representative of 2 experiments. e-f) Primary adipocytes were cultured in the absence or presence of M1-like or M2-like polarized SAT-derived macrophages in a transwell co-culture system (adipocytes, lower compartment; macrophages, upper compartment). The experiments were performed in the presence or absence of the α 4 -inh. or TNF-blocking antibody, as indicated. After 24 h of incubation, adipocyte VCAM-1 expression was analyzed by flow cytometry and is expressed as MFI units (n=4 separate primary cell isolations from SAT in e and n=5 separate primary cell isolations from SAT in f ). Data in ( e )-( f ) are pooled from 2 experiments. g) Correlation analysis between mRNA expression of UCP1 and VCAM1 in human SAT (n=61 human samples). h) Human subcutaneous pre-adipocytes were differentiated to adipocytes and 10 days after starting differentiation were treated without (control; Con) or with TNF or palmitate for 12 h. VCAM1 expression was analyzed and ACTB expression was used for normalization. VCAM1 expression of control-treated adipocytes was set as 1. Data are pooled from 4 experiments. i) Human inflammatory macrophages were allowed to adhere to TNF-pre-treated differentiated human adipocytes in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The percentage of adherent cells is shown (n=4 separate macrophage isolations). Data are representative of 2 experiments. j) Differentiated human adipocytes were treated with T3 and norepinephrine to induce a thermogenic response, washed and then co-cultured without or with human M1-like inflammatory macrophages (M1MΦ) in the same well (direct contact) for 1 h. Experiments were performed in the absence (control; Con) or presence of α 4 -inhibitor (α 4 -inh.). The expression of UCP1 in adipocytes (after exclusion of CD45-expressing cells) was detected; ACTB expression was used for normalization. UCP1 expression in adipocytes cultured in the absence of M1MΦ and in the absence of α 4 -inhibitor was set as 1. Shown is one representative experiment (n=4-5 cell culture treatments); similar results were observed in 2 experiments. Data are mean ± SEM.; *P < 0.05. Mann-Whitney U-test was used in ( a )-( f ) and ( h )-( j ) and one-tailed Pearson Correlation analysis in ( g ).

Article Snippet: Human VCAM1 and UCP1 expression were measured by quantitative real-time RT-PCR using the following probes: human VCAM1 (Hs01003370_m1, ThermoFisher Scientific), UCP1 (Hs00222453_m1, ThermoFisher Scientific).

Techniques: Cell Culture, Incubation, Control, Expressing, Flow Cytometry, Isolation, Derivative Assay, Co-Culture Assay, Blocking Assay, MANN-WHITNEY, One-tailed Test

Journal: Emerging Microbes & Infections

Article Title: Kyasanur Forest disease virus infection activates human vascular endothelial cells and monocyte-derived dendritic cells

doi: 10.1038/s41426-018-0177-z

Figure Lengend Snippet: a HDMECs were infected with KFDV at an MOI of 10. Mock-infected HDMECs were exposed to the same dose of UV-inactivated KFDV. At 24 and 48 h p.i., mRNA expression of E-selectin, ICAM1, and VCAM1 was analyzed by qRT-PCR and normalized to the expression of housekeeping genes. Fold increase indicates differences in normalized mRNA expression relative to expression in noninfected cells. b Flow cytometry was used to measure the protein levels of E-selectin, VCAM1, and ICAM1 expression in the KFDV-infected HDMECs at 48 h p.i. The production of cell-adhesion molecules is shown in overlapping histograms of relative fluoresce intensity in the analyzed cells; empty histograms with solid lines represent mock-infected (UV-inactivated virus) HDMECs, and gray histograms represent the KFDV-infected cells. c HDMECs were treated with TNF-α (positive control) or mock-infected with UV-inactivated KFDV or infected with KFDV at an MOI of 10 and incubated for 24 and 48 h. After incubation, the adhesion of BCECF–stained leukocytes was measured as described in the Materials and Methods. The leukocyte adhesion index represents the fold change relative to controls (y-axis). d HDMECs grown and fixed on slides at 48 h p.i. were stained with anti-flavivirus envelope (green) and anti-occludin (red) or anti-ZO-1 antibodies and counterstained with DAPI (blue). Bar = 20 µm. e Total RNA from uninfected, mock- and KFDV-infected HDMECs 48 h p.i. was used to determine the fold change in occludin and ZO-1 mRNA expression by qRT-PCR. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. h.p.i. = hours post infection

Article Snippet: PCR was performed using predeveloped TaqMan Assay Reagents [Assay IDs: ICAM-1 (Hs00164932_m1), VCAM-1 (Hs01003372_m1), SELE (Hs00174057_m1), occludin (Hs00170162_m1), and zonula occludens-1 (ZO-1/TJP1; Hs01551876_m1)] and TaqMan Gene Expression Master Mix (Applied Biosystems) on a LightCycler 480 II with a 96-well plate block (Roche, UK).

Techniques: Infection, Expressing, Quantitative RT-PCR, Flow Cytometry, Virus, Positive Control, Incubation, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Human and porcine aortic valve endothelial and interstitial cell isolation and characterization

doi: 10.3389/fcvm.2023.1151028

Figure Lengend Snippet: Human valvular endothelial cell (hVEC) isolation and characterization. ( A ) Workflow of VEC isolation: (1) Explanted aortic valve cusps were incubated for 10 min in collagenase type II solution. (2) VECs were carefully scraped with a scalpel into a dish filled with VEC medium, which (3) was transferred into a 15 ml tube and washed twice for 15 min at 1,000 rpm at room temperature. (4) Cells were incubated with CD105 magnetic beads for 15 min, and (5) the CD105 positive selection was carried out with MACS® separator, according to the manufacturer's protocol. (6) The specific VEC population was seeded into one well of a fibronectin-coated 6-well plate. ( B ) Gene expression of isolated hVECs and human valvular interstitial cells (hVICs) showed a positive expression of the endothelial markers von Willebrand factor ( vWF ), platelet adhesion molecule 1 ( PECAM1 ), endoglin ( CD105 ), and nitric oxide synthase 3 ( NOS3 ) in hVECs. ( C ) Brightfield and immunofluorescence images of VECs positive for vWF and CD31 and negative for alpha smooth muscle actin (α-SMA) and vimentin (VIM). ( D ) Migration analysis by scratch wound healing assay resulted in a scratch width of 15% after 8 h. Treatment of hVECs with H 2 O 2 for 24 h resulted in decreased ( E ) cell viability, increased ( F ) caspase 3/7 activity, and decreased ( G ) cell proliferation. ( H ) Life span of hVECs in cell culture at different passages (p). ( I ) Inducing EndMT by incubating VECs with tumor necrosis factor alpha (TNFα) for 7 days. TNFα led to an upregulation of α - SMA, VIM , cadherin 2 ( CDH2 ) and vascular adhesion molecule 1 ( VCAM1 ) and a downregulation of PECAM1, vWF and NOS3. ( B ) n = 5 donors, ( D ) n = 3 donors, ( E–I ) n = 3 donors with technical replicates indicated by one color per donor, * P < 0.05, ** P < 0.01, *** P < 0.001, analyzed by Student t -test, 2-tailed, unpaired, ( A ) created with BioRender.com.

Article Snippet: The following porcine TaqManTM primers were used: GAPDH (Ss03375629_u1), α-SMA (Ss04245588_m1), vWF (Ss04322692_m1), PECAM1 (Ss04322692_m1), VIM (Ss04330801_gH), CD105 (Ss03391353_m1), NOS3 (Ss03383840_u1), CDH2 (Ss06911356_m1), VCAM1 (Ss03390912_m1), ALPL (Ss06879568_m1), BMP2 (Ss03373798_g1), BGLAP (Ss03373655_s1), and SPP1 (Ss03391321_m1).

Techniques: Isolation, Incubation, Magnetic Beads, Selection, Gene Expression, Expressing, Immunofluorescence, Migration, Wound Healing Assay, Activity Assay, Cell Culture

Journal: Frontiers in Cardiovascular Medicine

Article Title: Human and porcine aortic valve endothelial and interstitial cell isolation and characterization

doi: 10.3389/fcvm.2023.1151028

Figure Lengend Snippet: Porcine valvular endothelial cell (pVECs) isolation and characterization. ( A ) Gene expression analysis of pVECs revealed a significant upregulation of von Willebrand factor ( vWF ), platelet adhesion molecule 1 ( PECAM1 ), endoglin ( CD105 ), nitric oxide synthase 3 ( NOS3 ), and vimentin ( VIM ) and a downregulation of alpha smooth muscle actin ( α-SMA ) and cadherin 2 ( CDH2 ) compared to porcine valvular interstitial cells (pVICs). ( B ) Brightfield images and immunofluorescence staining of pVECs showing a positive signal for vWF and an absent signal for α-SMA. ( C ) Analysis of migration properties by scratch wound healing assay revealed a scratch closure to 70%. pVECs treated with 100 µM H 2 O 2 showed a decrease in ( D ) cell viability, ( E ) caspase 3/7 activity, and ( F ) cell proliferation. ( G ) Life span of pVECs in cell culture at different passages (p). ( H ) In vitro EndMT induction by TNFα for 7 days. TNFα displayed downregulated endothelial marker expression, ( PECAM1, vWF, NOS3 ) and mesenchymal markers, ( α-SMA, VIM, CDH2 ). CDH2 and vascular cell adhesion molecule 1 ( VCAM1 ) was upregulated. ( A ) n = 5 donors, ( C ) n = 3 donors, ( D–H ) n = 3 donors with technical replicates indicated by one color per donor, * P < 0.05, ** P < 0.01, *** P < 0.001, analyzed by Student t -test, 2-tailed, unpaired, (a) created with BioRender.com.

Article Snippet: The following porcine TaqManTM primers were used: GAPDH (Ss03375629_u1), α-SMA (Ss04245588_m1), vWF (Ss04322692_m1), PECAM1 (Ss04322692_m1), VIM (Ss04330801_gH), CD105 (Ss03391353_m1), NOS3 (Ss03383840_u1), CDH2 (Ss06911356_m1), VCAM1 (Ss03390912_m1), ALPL (Ss06879568_m1), BMP2 (Ss03373798_g1), BGLAP (Ss03373655_s1), and SPP1 (Ss03391321_m1).

Techniques: Isolation, Gene Expression, Immunofluorescence, Staining, Migration, Wound Healing Assay, Activity Assay, Cell Culture, In Vitro, Marker, Expressing

Journal: Vascular Cell

Article Title: A novel endothelial-derived anti-inflammatory activity significantly inhibits spontaneous choroidal neovascularisation in a mouse model

doi: 10.1186/s13221-016-0036-4

Figure Lengend Snippet: Inhibition of TNFα-induced expression of E-selectin and VCAM-1 by ECPCM. a HAEC were treated with TNFα for 2 h in collection medium or ECPCM, then relative gene expression levels normalized to the collection media control were determined by real-time PCR analysis. Percentage inhibition of gene expression by ECPCM was calculated by comparing treatment with ECPCM and TNFα to treatment with TNFα in collection medium. p -value: ** <0.01 compared to TNFα in collection medium. Data = mean ± SEM. b HAEC were treated for 6 h with or without TNFα in collection medium or ECPCM. Western blot analysis was performed with anti-VCAM-1, anti-E-selectin and anti-β-actin antibodies. The image is representative of three separate experiments. c Relative density quantification of western blot bands for TNFα-treated HAEC in panel ( b ), performed using ImageJ software. E-selectin and VCAM-1 bands were normalised to the β-actin loading control band. Data = mean ± SEM; n = 3 per treatment. p -value: * <0.05 compared to TNFα in collection medium. d U937 cells were stained with calcein AM and then placed on top of HAEC for 30 min. After three rinses with PBS, U937 attachment to EC was determined by counting the fluorescent cells. Changes in the number of attached cells are expressed as fold changes over the control (treatment in collection medium). p -value: ** <0.01 compared to TNFα in collection medium. Data = Mean ± SEM. e Representative images of U937 cells stained with calcein AM and attached to cytokine-activated HAEC treated in collection medium or ECPCM. Black background = HAEC; white dots = fluorescent U937 cells attached to the HAEC

Article Snippet: Quantitative real-time PCR was performed using specific human TaqMan probes (Applied Biosystems, Foster City, CA) for E-selectin (Hs00950401_m1) and VCAM-1 (Hs01003369_m1).

Techniques: Inhibition, Expressing, Gene Expression, Control, Real-time Polymerase Chain Reaction, Western Blot, Software, Staining

Journal: Vascular Cell

Article Title: A novel endothelial-derived anti-inflammatory activity significantly inhibits spontaneous choroidal neovascularisation in a mouse model

doi: 10.1186/s13221-016-0036-4

Figure Lengend Snippet: ECPCM treatment does not affect mRNA stability of E-selectin or VCAM-1 transcripts. HAEC were treated with TNFα in collection medium for 2 h. The medium was then changed to collection medium + TNFα ( dotted line ), collection medium + TNFα + actinomycin D ( black line ) or ECPCM + TNFα + actinomycin D ( grey line ). Transcript levels were assessed by real-time PCR after 30 min, 1, 2 and 4 h of treatment. The mRNA stability for both genes did not change upon ECPCM treatment. Data = mean ± SEM

Article Snippet: Quantitative real-time PCR was performed using specific human TaqMan probes (Applied Biosystems, Foster City, CA) for E-selectin (Hs00950401_m1) and VCAM-1 (Hs01003369_m1).

Techniques: Real-time Polymerase Chain Reaction

Journal: Vascular Cell

Article Title: A novel endothelial-derived anti-inflammatory activity significantly inhibits spontaneous choroidal neovascularisation in a mouse model

doi: 10.1186/s13221-016-0036-4

Figure Lengend Snippet: ECPCM significantly decreases binding of p65 to E-selectin and VCAM-1 promoters upon TNFα treatment. HUVEC were treated for 1 h with or without TNFα in collection medium or ECPCM. The extracted chromatin was immunoprecipitated using anti-p65 antibody or isotype-matched IgG as control. The immunoprecipitated DNA was then quantified using real-time PCR. The level of p65 binding to the specific promoters was expressed as fold-change over the IgG control. p -value: * <0.01, ** <0.001 comparing ECPCM + TNFα to collection medium + TNFα treatment. Data = mean ± SEM

Article Snippet: Quantitative real-time PCR was performed using specific human TaqMan probes (Applied Biosystems, Foster City, CA) for E-selectin (Hs00950401_m1) and VCAM-1 (Hs01003369_m1).

Techniques: Binding Assay, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction

Journal: Vascular Cell

Article Title: A novel endothelial-derived anti-inflammatory activity significantly inhibits spontaneous choroidal neovascularisation in a mouse model

doi: 10.1186/s13221-016-0036-4

Figure Lengend Snippet: a Dose-dependent anti-inflammatory effects of ECPCM. HAEC were treated for 2 h with TNFα in collection medium, ECPCM, or serial dilutions (1:2) of ECPCM in collection medium. The graph shows the percentage of expression of E-selectin and VCAM-1 relative to that observed for TNFα in collection medium, as determined by real-time PCR analysis. Data = mean ± SEM. b ECPCM anti-inflammatory activity is not affected by proteinase K or RNase treatment. Gene expression of E-selectin and VCAM-1 was analysed by real-time PCR after treatment for 2 h with TNFα. HAEC were treated with collection medium, ECPCM or ECPCM previously treated with proteinase K or RNase. p value: * <0.001 compared to treatment with TNFα in collection medium. Data = mean ± SEM

Article Snippet: Quantitative real-time PCR was performed using specific human TaqMan probes (Applied Biosystems, Foster City, CA) for E-selectin (Hs00950401_m1) and VCAM-1 (Hs01003369_m1).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Gene Expression