c-mvec  (Lonza)

Journal: Annals of the Rheumatic Diseases

Article Title: Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

doi: 10.1136/annrheumdis-2017-212120

Figure Lengend Snippet: Suppression of angiogenesis in an organotypic co-culture assay by systemic sclerosis (SSc) fibroblasts is reversed by pigment epithelium-derived factor (PEDF) knockdown. (A, C) Images show representative microscopic fields from co-culture assays of human dermal microvascular endothelial cells (MVECs) (A) or human umbilical vein endothelial cells (HUVECs) (C) seeded onto confluent fibroblasts (FBs), healthy control (HC-FBs) or SSc (SSc-FBs), stained for the endothelial marker CD31 (fibroblasts are seen unstained in the background). Note that HUVECs reproduce the behaviour of MVECs in the organotypic assays. (B, D) Histograms show the number of tubules and total tubule length quantified using Angiosys software, represented as mean±SEM (n=12 microscopic fields at ×4 magnification from triplicate wells). (E) Representative western blot showing intracellular PEDF levels in SSc fibroblasts treated with GolgiPlug, non-silencing control (NS) or with PEDF depletion (shPEDF) by means of lentiviral short-hairpin RNA (sh). (F) Images show representative microscopic fields from co-culture assays of HUVECs seeded onto confluent SSc fibroblasts (SSc-FBs), non-silencing control (NS) or with PEDF depletion (shPEDF). (G) Quantification of the number of tubules and total tubule length represented as mean±SEM (n=12 microscopic fields at ×4 magnification from triplicate wells). **P<0.01, ***P<0.001 by unpaired t-test. Scale bars, 100 µm.

Article Snippet: Human dermal microvascular endothelial cells (MVECs) were purchased from PromoCell, UK, cultured in Endothelial Cell Growth Medium BulletKit (Lonza, Slough, UK) and used at passages 2–4.

Techniques: Co-culture Assay, Derivative Assay, Co-Culture Assay, Staining, Marker, Software, Western Blot, shRNA

Journal: The Journal of Immunology Author Choice

Article Title: Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions

doi: 10.4049/jimmunol.1600823

Figure Lengend Snippet: Endothelial MAPK activation in response to ICAM-1 ligation and cross-linking. ( A ) GPNT were subjected to ICAM-1 cross-linking (XL), with secondary clustering, for the indicated length of time and MAPK phosphorylation analyzed. Representative results and quantification of kinase activation (normalized mean ± SEM) from three independent experiments are shown. ( B ) Representative analysis of MAPK phosphorylation following ICAM-1 ligation (without secondary clustering) for the indicated times and densitometric quantification of kinase activation of three independent experiments. MAPK levels were not significantly affected by ICAM-1 ligation or cross-linking . ( C ) Postconfluent, serum-starved GPNT cells were either left untreated or incubated with 5 μg/ml anti–ICAM-1 (1A29) or isotype-matched control IgG for 10 min. ( D – F ) Primary rat brain MVEC (D) or hCMEC/D3 (E) or human dermal MVEC (F) were stimulated by ICAM-1 cross-linking (XL) or ICAM-1 ligation for the indicated times and MAPK phosphorylation analyzed as described in (B). Data were compared with the corresponding time 0 controls and significant differences are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human dermal MVEC (PromoCell) were cultured on tissue culture plasticware coated with gelatin (Sigma) and maintained in EC growth media MV2 (PromoCell).

Techniques: Activation Assay, Ligation, Incubation

Journal: The Journal of Immunology Author Choice

Article Title: Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions

doi: 10.4049/jimmunol.1600823

Figure Lengend Snippet: Role of MAPK in ICAM-1–mediated gene expression. ( A ) Total RNA was isolated from untreated (NT) or 4 h ICAM-1 cross-linked (XL) GPNTs and analyzed by semiquantitative RT-PCR for message levels of VCAM-1, TNF-α, COX-2, CCL2, ICAM-1, and GAPDH. ( B and C ) Confluent hCMEC/D3 (B) or human dermal MVEC (hDMEC) (C) were either left untreated or subjected to ICAM-1 cross-linking. At the indicated times TNF-α concentration in the culture supernatant was determined by ELISA. Shown are mean levels ± SEM of TNF-α above those in control cells from three independent experiments. ( D and E ) hCMEC/D3 (D) or human dermal MVEC (hDMEC) (E) were left untreated (NT) or subjected to ICAM-1 cross-linking (XL) for 24 h. The concentration of CXCL8, CXCL10, CCL2, CCL3, and CCL4 in the supernatant was measured by multianalyte flow assay. Where indicated anti–TNF-α (1 μg/ml) was included during the stimulation period to determine if altered chemokine secretion was a consequence of TNF-α induction. Shown are mean concentrations ± SEM of chemokines in the culture supernatant as determined from three independent experiments. ( F ) Confluent GPNT ECs were either left untreated (NT) or treated with 200 U/ml TNF-α for 12 h or subjected to ICAM-1 cross-linking (XL) for 12 h prior to immunoblot analysis of VCAM-1 and tubulin. Shown is a representative blot and densitometric quantification of three independent experiments. Where indicated cross-linking was performed in the presence of 50 μM U0126, SP600125, or SB202190. White separation lines indicate where lanes from the same blots were joined. ( G ) GPNT ECs were transiently nucleofected with pVCAM-1-luc, reseeded and allowed to grow to confluence (48–72 h posttransfection), and then subjected to ICAM-1 cross-linking (XL) for 6 h or stimulation with IL-1β (IL1β). Where indicated cross-linking was performed in the presence of 50 μM U0126, SP600125, or SB202190. Mean ± SEM luciferase activity (relative to untreated cells) was determined from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human dermal MVEC (PromoCell) were cultured on tissue culture plasticware coated with gelatin (Sigma) and maintained in EC growth media MV2 (PromoCell).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Luciferase, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions

doi: 10.4049/jimmunol.1600823

Figure Lengend Snippet: Endothelial JNK regulates lymphocyte TEM. ( A ) GPNT monolayers were pretreated or not with actinomycin D (Act D, 5 μg/ml) and subsequent TEM of Th1 lymphocytes (PAS, see also ) measured after 30 min. Whereas Act D did not affect 30 min TEM rates, it inhibited all subsequent TEM events (measured up until 4 h). ( B ) TEM assay as in (A) with the exception that GPNT monolayers were left untreated (NT) or treated with 50 μM U0126 (U0), SP600125 (SP) or SB202190 (SB) for 1 h prior to a 30 min TEM assay. Due to the high washout rate of U0126 from GPNT cells (see ), TEM experiments were also conducted with U0126 present throughout. However, even under these conditions TEM was not inhibited (data not shown). ( C ) TEM assay as in (B) except that primary rat brain MVEC were either left untreated (NT) or pretreated with 50 μM SP600125 for 1 h prior to addition of Ag-specific T lymphocytes. ( D ) TEM assay as in (B) with the exception that GPNT were either left untreated (NT) or treated with 1 μM L-JNKi for 1 h prior to the addition of T lymphocytes. ( E ) TEM assay as in (B) except that GPNT cells were transfected with wild-type (WT) or dominant-negative (DN) JNK1, JNK2, or MKK7 48 h before TEM and adhesion were analyzed. ( F and G ) TEM assay as in (B) except that TEM of human CD4 + cells across hCMEC/D3 (F) or human dermal MVEC (G) was measured following EC pretreatment with 50 μM U0126 (U0), SP600125 (SP), or SB202190 (SB) for 1 h. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human dermal MVEC (PromoCell) were cultured on tissue culture plasticware coated with gelatin (Sigma) and maintained in EC growth media MV2 (PromoCell).

Techniques: Transfection, Dominant Negative Mutation