Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 4. EndT and EndMT of Metastatic Melanoma Cells in Mouse (A) Whole-mount staining of pulmonary artery with GFP+ cells located at the endothelium (Videos S1, S2, and S3). Bars: 10 mm. (B and C) EndT occurred in lymph nodes. EndT occurred in LYVE-1+ lymphatic vessles (B) and CD31+ blood vessels (C) in lymph nodes. Bars: 20 mm. (D) LYVE-1 immunostaining on lung cryosections. Bar: 20 mm. (E–J) Immunostaining indicated EndT is transient during tumor progression. At primary tumor sites, GFP+ cells were CD31-, VE-cadherin+ (E and H), while in the lung blood vessels, GFP+ cells were both CD31+ and VE-cadherin+ (F and I). GFP+ cells lost both endothelial markers when populating metastases in the lung (G and J). Bars: 20 mm. (K) CD31+/a-SMA+/GFP+ cell (arrowhead) inside the alveolar capillary. CD31+/aSMA+/GFP cell (arrow) near the GFP+ cell showed the same phenotype. It is possible that this is an authentic vascular endothelial cell undergoing EndMT or a metastatic cell that was not labeled with GFP. Bars: 10 mm. (A–K) Mice, n R 3. (L) Correlation between the number of GFP+ cells per vessel and the number of metastasis foci or the size of metastasis foci per mouse. The number of GFP+ cells/vessel vs. number of metastasis foci, r = 0.7950, p = 0.03*; number of GFP+ cells/vessel vs. metastasis size, r = 0.8012, *p = 0.03. Mice, n = 7.

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: Staining, Immunostaining, Labeling

Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 5. In Vitro System of Melanoma Cell EndT (A–C) Melanoma/endothelial marker immunostaining of Q-YUWERA cells (cells with green dots) cultured alone (A, VE-cadherin showed background staining in nuclei) or 5 days after co-culturing with PAECs (B and C). CD31 and HMB45 double staining in (B), VE-cadherin and HMB45 double staining in (C). Bars: 20 mm. Independent experiments, n R 3. (D) Imaging flow cytometry confirmed the expression of CD31 by individual YUWERA (APC+) cells after the co-culture. BF, bright field; SSC, side scatter; APC, CellTrace far red-labeled YUWERA cells. (E) YUWERA cells were labeled with a CFSE proliferation kit and co-cultured with PAECs for 5 days with 300 nM sunitinib treatment or control solution. Cells were subjected to CD31 staining followed by flow cytometry analysis. Independent experiments, n = 3; replicates, n = 3 in each experiment. 300 nM sunitinib treatment significantly increased the percentage of CD31+ YUWERA cells of the overall YUWERA cells in the co-culture (control group 2.878% ± 0.1309%, sunitinib group 4.677% ± 0.3125%,*p = 0.0357, shown as mean ± SEM by a Mann-Whitney test).

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: In Vitro, Marker, Immunostaining, Cell Culture, Staining, Double Staining, Imaging, Cytometry, Expressing, Co-Culture Assay, Labeling, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 6. EndT Inferred from Single-Cell RNA-Seq Data and Immunofluorescence in Metastatic Human Melanoma Biopsies (A) tSNE plot from single-cell RNA-seq showing metastatic melanoma cells of different patients (23). Malignant melanoma cells were selected based on aberrant copy number profiles, which are mutually exclusive from authentic endothelial cells, and plotted based on their pigmentation activity score, based on AUCell (37). The zoom shows cells of a melanoma patient that does not show pigmentation activity but had rare cells with high expressional activity for an endothelial gene signature. (B–I) Immunofluorescence on metastatic melanoma biopsies from BRAFV600E-harboring patients. (B and C) Representative double-immunofluorescence mi- crographs for BRAFV600E and CD31 of metastatic melanoma biopsies in the lung (B) and in the brain (C). (D and E) BRAFV600E+/CD31+ cells (arrows) localized inside the vasculature of metastatic melanoma biopsies in the lung (D) and in the brain (E). Bars: 50 mm. (F and G) Representative triple-immunofluorescence micrographs for BRAFV600E, MITF and CD31 of metastatic melanoma biopsies in the lung F) and in the brain (G). (H and I) BRAFV600E+/CD31+ cells inside the vasculature of metastatic melanoma biopsies in the lung (H) and in the brain (I) were negative for melanocytic marker MITF (arrows). Bars: 50 mm.

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: RNA Sequencing, Activity Assay, Marker

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Endothelial cells characterization regarding morphological, immunophenotyping, and vessel-like structures assay. ( A ) Phase contrast micrography demonstrating the polygonal morphology of aortic artery endothelial cells (PAEC), coronary artery endothelial cells (CAEC), human umbilical vein endothelial cells (HUVEC), and pulmonary artery endothelial cells (HPAEC) cells (100× magnification). ( B ) Immunophenotyping of ECs by flow cytometry. ( C ) All endothelial cells (PAEC, CAEC, HUVEC, and HPAEC) were able to form vessel-like structures when grown in matrigel, evidencing characteristics typical of CEs (40× and 100× magnification).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Characterization of EndMT induction by TGF-β2 (10 ng/mL) in cell lines ( A ) PAEC, ( B ) CAEC, ( C ) HPAEC, and ( D ) HUVECs (non-treated or treated with TGF-β2). Immunofluorescence microscopy of cell lines induced to EndMT shows a decrease in the fluorescent intensity of CD31 (green) in PAECs, CAECs, and HUVECs cells. The nuclei were stained with DAPI (blue) and F-actin were stained with Phalloidin (red) (scale bar 50 µM; representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: TGF-β2 decrease formation of vessel-like structures in the cell lines (CAEC, PAEC, HPAEC, and HUVEC). The cells were treated with TGF-β2 and evaluated the capacity formation of vessel-like structures. This inhibitory effect was observed mainly in PAECs (representative image of one replicate; n = 3).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Effect of EndMT on the activation of the Erk pathway. The cells (CAEC, PAEC, HUVEC and HPAEC) were cultured for five days in presence TGF-β2 (10 ng/mL). Aliquots were withdrawn after the treatment and evaluated by ( A ) Multiplex technique analysis Array Kit ( n = 3, * p ≤ 0.05) and ( B ) western blotting using phospho-Erk1/2 (Thr202/Tyr204) and ERK1/2. β-actin were used as endogenous controls (representative image of one replicate of each sample). ( C ) Chemical inhibitor against MEK1/2 (U0126; 1 μM) inhibits the increase of ERK1/2 phosphorylation in the PAECs treated with TGF-β2. 1) U0126; 2) U0126-15′ TGF-β2; 3) U0126-30′ TGF-β2; 4) TGF-β2-15′; 5) TGF-β2-30′. GAPDH were used as endogenous controls (representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Activation Assay, Cell Culture, Multiplex Assay, Western Blot, Phospho-proteomics

Journal: The Journal of Biological Chemistry

Article Title: Novel Peptide for Attenuation of Hyperoxia-induced Disruption of Lung Endothelial Barrier and Pulmonary Edema via Modulating Peroxynitrite Formation *

doi: 10.1074/jbc.M114.585356

Figure Lengend Snippet: l-NAME attenuates hyperoxia-induced disruption of endothelial monolayer barrier integrity. PAECs were treated with and without l-NAME (3 mm) and exposed to hyperoxia for 48 h. TEER was continuously monitored as described under “Experimental Procedures.” A, changes in TEER of endothelial monolayer under normoxia and hyperoxia. B, changes in TEER of endothelial monolayer under hyperoxia with and without l-NAME. Results are expressed as means ± S.E.; n = 4. *, p < 0.05 versus normoxia; #, p < 0.05 versus l-NAME+normoxia; **, p < 0.05 versus control hyperoxia.

Article Snippet: Primary bovine pulmonary artery endothelial cells (PAECs) were obtained from Cell Applications (San Diego, CA).

Techniques: Disruption, Control

Journal: The Journal of Biological Chemistry

Article Title: Novel Peptide for Attenuation of Hyperoxia-induced Disruption of Lung Endothelial Barrier and Pulmonary Edema via Modulating Peroxynitrite Formation *

doi: 10.1074/jbc.M114.585356

Figure Lengend Snippet: Uric acid prevents hyperoxia-induced disruption of lung endothelial barrier in the second phase and apoptosis. PAECs were treated with and without uric acid (3 mm) and exposed to hyperoxia for 48 h, during which TEER was continuously monitored. After exposure, apoptotic cells were detected using TUNEL assay as described under “Experimental Procedures.” A, changes in TEER of endothelial monolayer. B, alterations in the numbers of TUNEL-positive cells. Results are expressed as means ± S.E.; n = 4. *, p < 0.05 versus normoxia; #, p < 0.05 versus UA+normoxia; **, p < 0.05 versus hyperoxia. UA = uric acid.

Article Snippet: Primary bovine pulmonary artery endothelial cells (PAECs) were obtained from Cell Applications (San Diego, CA).

Techniques: Disruption, TUNEL Assay

Journal: The Journal of Biological Chemistry

Article Title: Novel Peptide for Attenuation of Hyperoxia-induced Disruption of Lung Endothelial Barrier and Pulmonary Edema via Modulating Peroxynitrite Formation *

doi: 10.1074/jbc.M114.585356

Figure Lengend Snippet: Peptide P326TAT attenuates hyperoxia-induced disruption of endothelial monolayer barrier integrity. PAECs were treated with peptide P326TAT (20 μm) or control peptide PlwTAT (20 μm) and exposed to hyperoxia for 48 h. TEER was continuously monitored as described under “Experimental Procedures.” A, changes in TEER of endothelial monolayer under hyperoxia with control peptide PlwTAT. B, changes in TEER of endothelial monolayer under hyperoxia with P326TAT. Results are expressed as means ± S.E.; n = 4. *, p < 0.05 versus normoxia; #, p < 0.05 versus P326TAT+normoxia or PlwTAT+normoxia; **, p < 0.05 versus hyperoxia.

Article Snippet: Primary bovine pulmonary artery endothelial cells (PAECs) were obtained from Cell Applications (San Diego, CA).

Techniques: Disruption, Control

Journal: The Journal of Biological Chemistry

Article Title: Novel Peptide for Attenuation of Hyperoxia-induced Disruption of Lung Endothelial Barrier and Pulmonary Edema via Modulating Peroxynitrite Formation *

doi: 10.1074/jbc.M114.585356

Figure Lengend Snippet: Peptide P326TAT attenuates hyperoxia-induced apoptosis of lung endothelial cells. PAECs were exposed to hyperoxia or normoxia in the presence of P326TAT (20 μm) or control peptide PlwTAT (20 μm) for 48 h, and then apoptosis was evaluated using TUNEL assay. A, representative images of TUNEL staining. WO peptide, without peptide. B, bar graph depicting the changes in the numbers of TUNEL-positive cells. Results are expressed as means ± S.E.; n = 4. *, p < 0.05 versus normoxia; #, p < 0.05 versus PlwTAT+hyperoxia or hyperoxia only.

Article Snippet: Primary bovine pulmonary artery endothelial cells (PAECs) were obtained from Cell Applications (San Diego, CA).

Techniques: Control, TUNEL Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Novel Peptide for Attenuation of Hyperoxia-induced Disruption of Lung Endothelial Barrier and Pulmonary Edema via Modulating Peroxynitrite Formation *

doi: 10.1074/jbc.M114.585356

Figure Lengend Snippet: Peptide P326TAT prevents both caspase-dependent and caspase-independent apoptosis of lung endothelial cells. PAECs were treated with peptide P326TAT (20 μm) or control peptide PlwTAT (20 μm) and exposed to hyperoxia for 48 h, after which caspase-3 activity and AIF protein level in the nuclear fraction were measured as described under “Experimental Procedures.” A, changes in caspase-3 activity. B, representative image of Western blot of AIF. WO peptide, without peptide. C, bar graph depicting changes in nuclear AIF levels of PAECs. Results are expressed as means ± S.E.; n = 4. *, p < 0.05 versus normoxia.

Article Snippet: Primary bovine pulmonary artery endothelial cells (PAECs) were obtained from Cell Applications (San Diego, CA).

Techniques: Control, Activity Assay, Western Blot