Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: Juxtamembrane 2 (JM2) is not cytotoxic. Human microvascular endothelial cells (HMVECs) were treated with JM2 at 12.5, 25, 50, 100, and 200 μM concentrations for 2 h at 37°C, 5% CO2. The media were then sampled for lactate dehydrogenase (LDH) as a marker for cell death. No significant differences were observed between control (No Peptide) and any concentration of JM2. n = 3, with each n representing the average of 3 replicates; error bars represent SE.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Marker, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 specifically enhances connexin43 (Cx43)-β-tubulin interaction. A and B: Cx43-HeLa cell lysates were incubated with β-tubulin-glutathione S-transferase (GST) bound to glutathione-Sepharose beads plus vehicle (H2O; Veh) or 25 μM JM2. Eluted proteins were analyzed by immunoblotting for Cx43 (A) or β-tubulin (B). β-Tubulin pulled down Cx43 in control conditions (vehicle), and this was increased in the presence of JM2 (A, “β-tubulin Pull-Down”), indicating that JM2 specifically enhances the interaction between Cx43 and β-tubulin. This was supported by the observation that the input amounts of Cx43 and β-tubulin subjected to pull-down were equivalent between Veh and JM2 treatments (A and B “Input” blots, respectively) and that JM2 did not affect the amount of β-tubulin pulled down by a β-tubulin antibody (B, “β-tubulin Pull-Down”). C: HMVECs were treated for 2 h with either vehicle (H2O; “Veh”), 50 μM JM2, or 50 μM control peptide (CP). Cells were fixed and labeled for Cx43-β-tubulin interaction (green), and the nucleus (blue). Differential interference contrast (DIC; gray scale) was used to approximate cell-cell borders (dashed lines). Boxed regions indicate the location of the enlarged insets. Scale bar = 10 μm. D: Cx43-β-tubulin interaction was quantified as the number of Duolink signals detected per cell. The number of signals detected in JM2-treated cells was significantly greater than in vehicle (Veh) or control peptide (CP). n = 3, with each n representing the average of 5 replicates; error bars represent SD. Nonlinear level adjustments were applied to Western blot images to enhance visibility. ****P < 0.001.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Incubation, Western Blot, Labeling

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 decreases gap junction (GJ) size while increasing labeling for microtubules. HMVECs were treated for 2 h with either vehicle (H2O; “Veh”) or 50 μM JM2. A: cells were fixed and labeled for Cx43 (cyan), α-tubulin (yellow), and the nucleus (magenta); Scale bar = 10 μm. B: total Cx43 fluorescence was unaffected by JM2. C and D: similarly, GJ number was measured and found to be unaffected by JM2 (C), while there was a significant decrease in the level of cell border-associated Cx43 fluorescence (D). E: consistent with the reduction in cell border Cx43 fluorescence, GJ size was reduced. F: concomitantly, the level of intracellular Cx43 fluorescence was significantly increased in JM2-treated cells. G: microtubule fluorescence was also significantly increased by JM2 treatment. For all graphs, n = 3, with each n representing the average of 5 replicates; error bars represent SD. *P < 0.05, **P < 0.01.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Labeling, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 increases the accumulation of Cx43-containing secretory vesicles. HMVECs were treated for 2 h with either vehicle (H2O; “Veh”) or 50 μM JM2. A: cells were fixed and labeled for Cx43 (red), the trans-Golgi network (TGN) protein TGN38 (green), and the nucleus (blue). Arrows indicate a number of puncta with Cx43 TGN38 colocalization that are likely secretory vesicles; Scale bar = 10 μm. B–D: colocalization of Cx43 and TGN38 was significantly increased by JM2 as assessed by Pearson’s coefficient (B), the amount of colocalized pixels as a % of total Cx43 pixels (C), and the amount of colocalized pixels as a % of total TGN38 pixels (D); n = 3, with each n representing the average of 3 replicates; error bars represent SD. *P < 0.05, **P < 0.01.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Labeling

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: JM2 treatment inhibits both gap junction intercellular communication and hemichannel function. A: HMVECs were treated with either vehicle (H2O and EtOH), 50 μM JM2, or 50 μM flufenamic acid (FFA) for 2 h, and calcein-AM was loaded during the final 30 min. Selected cells (arrows) were then photobleached by high-intensity laser light and recovery was monitored. Scale bar = 20 μm. B: average fluorescence readings were plotted over time. n = 3, with each n representing the average of 3 replicates; error bars represent SE. C and D: nonlinear regression to an exponential decay function was used to determine the maximum predicted recovery (F∞; C) and the recovery rate constant (k; D). Error bars represent SD. E: connexin43 (Cx43) and and wild-type (WT) HeLa cells were treated with either vehicle (H2O and EtOH; “Veh”), 50 μM JM2, 50 μM FFA, or 5 μM mefloquine (MFQ) for 2 h, then exposed to ethidium bromide (EtBr) for 15 min. Cells were fixed and imaged, and the quantified relative fluorescence is displayed. Dashed line indicates the level of autofluorescence in Cx43-HeLa cells. F: HMVECs were treated, imaged, and quantified as in E. For graphs in E and F, n = 4, with each n representing the average of 5 replicates; error bars represent SD. *P < 0.05, **P < 0.01, ***P< 0.001, ****P < 0.001.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Mechanism of action of the anti-inflammatory connexin43 mimetic peptide JM2

doi: 10.1152/ajpcell.00229.2016

Figure Lengend Snippet: A control peptide does not affect hemichannel function. HMVECs were treated with either vehicle (H2O and EtOH; “Veh”), 50 μM control peptide (CP), or 50 μM FFA for 2 h, then exposed to EtBr for 15 min. Cells were fixed and imaged, and the quantified relative fluorescence is displayed. No significant difference was observed between vehicle control and control peptide treatment conditions; n = 3, with each n representing the average of 5 replicates; error bars represent SD. *P < 0.05.

Article Snippet: Human microvascular endothelial cells (HMVECs; Lonza, Allendale, NJ) were cultured in EGM-2MV medium (Lonza) according to manufacturer instructions and used for experiments at passage 5 or below.

Techniques: Fluorescence

Journal: Cancers

Article Title: Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells

doi: 10.3390/cancers12102850

Figure Lengend Snippet: Effects of PDIA1 inhibition on wound-healing and migration of breast cancer cells and endothelial cells. ECIS Wound-Healing Assay of hLMVEC, MCF-7 and MDA-MB-231 cells and breast cancer sublines with silencing of PDIA1 (shN, shPDIA1-1 and shPDIA1-3). Real time tracings in hLMVECs ( A ), MCF-7 ( C ) and MDA-MB-231 ( E ) cell lines after addition of bepristat 2a at various concentrations (1, 10, 30 or 50 µM) and after PDIA1 silencing in MCF-7 ( G ) and MDA-MB-231 ( I ) cell lines. Area under the curve boxplots represent AUC quantitation of changes in migration rate of bepristat 2a-treated hLMVECs ( B ), MCF-7 ( D ) and MDA-MB-231 ( F ) cell lines versus non-treated controls as well as MCF-7 ( H ) or MDA-MB-231 ( J ) sublines transduced against PDIA1 (shPDIA1-1, shPDIA1-3) or wild type cells regarding to negative sequence (shN). The line graphs and AUC boxplots represent mean ± SD of three independent experiments. Statistical analysis was calculated using parametric one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Article Snippet: Human lung microvascular endothelial cell line (hLMVECs) was obtained from Cell Applications (San Diego, CA, USA).

Techniques: Inhibition, Migration, Wound Healing Assay, Quantitation Assay, Sequencing

Journal: Cancers

Article Title: Protein Disulphide Isomerase A1 Is Involved in the Regulation of Breast Cancer Cell Adhesion and Transmigration via Lung Microvascular Endothelial Cells

doi: 10.3390/cancers12102850

Figure Lengend Snippet: Effects of exogenous PDIA1 and PDIA3 on adhesive interaction between breast cancer cells and different substrates. Effect of exogenous proteins PDIA1 and PDIA3 on adhesion of MCF-7 ( A – F ) and MDA-MB-231 ( G – L ) cells to collagen type I, fibronectin and lung microvascular hLMVEC cells, respectively. Data represent mean ± SD of three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (* p = 0.05, ** p = 0.01, *** p = 0.001).

Article Snippet: Human lung microvascular endothelial cell line (hLMVECs) was obtained from Cell Applications (San Diego, CA, USA).

Techniques: Adhesive

Journal: Tissue Engineering and Regenerative Medicine

Article Title: A Novel Hypothesis and Characterization to Isolate Microvascular Endothelial Cells Simultaneously with Adipose-Derived Stem Cells from the Human Adipose-Derived Stromal Vascular Fraction

doi: 10.1007/s13770-021-00332-5

Figure Lengend Snippet: Comparative analysis of properties according to the HMVEC-A isolation method. A Morphology and tube formation of HMVEC-A isolated by the vascular fraction (VF) isolation, magnetic-activated cell sorting (MACS) isolation method, and fluorescence-activated cell sorting (FACS) isolation methods were compared with Lonza HUVEC and HMVEC by phase-contrast microscopy; magnification, 40 × . B The VF isolation method produced a significantly higher yield than the FACS isolation method and MACS isolation method (*p < 0.05, **p < 0.01, n = 3). The doubling time was in the order of VF isolation method, MACS isolation method and FACS isolation method, and VF showed the fastest doubling time, but there was no statistically significant difference. The error bars indicate the standard errors of the mean of triplicate measurements. C FACS verification of EC-specific marker expression according to each isolation method. Positivity for the endothelial-specific cell surface antigens CD31 (PECAM1) and CD146 (MCAM) is indicated. Cells were analyzed at passage 2 (n = 3)

Article Snippet: Proliferation assays The proliferation of HMVEC-As isolated by three methods, Lonza HUVECs and Lonza HMVECs was assessed with a solution containing 10% Cell Counting Kit-8 reagent (Dojindo, Kumamoto, Japan).

Techniques: Isolation, FACS, Fluorescence, Microscopy, Produced, Marker, Expressing

Journal: Blood

Article Title: Netrin-4 induces lymphangiogenesis in vivo

doi: 10.1182/blood-2009-11-252338

Figure Lengend Snippet: Netrin-4 induces HMVEC-dLy proliferation, migration, tube formation and survival. (A) Mitogenic potential of different doses of Netrin-4 on lymphatic dermal human microvascular endothelial cells (HMVEC-dLys) compared with several known lymphangiogenic growth factors (FGF-2 or bFGF, HGF, VEGF-A (VA), VEGF-C (VC), and complete (CM) or basal cell (BM) culture media. Cell number was assessed using dojindo reagent 72 hours after treatment and expressed as fold increase versus control. (B) HMVEC-dLy proliferation under a single dose of Netrin-4 (500 ng/mL), FGF-2 (40 ng/mL), VEGF-A (50 ng/mL), or VEGF-C (300 ng/mL) assessed every 24 hours for 3 days. (C) Chemotactic effects of different doses of Netrin-4 and VEGF-C on HMVEC-dLys in a Boyden chamber assay. (D) HMVEC-dLy adhesion on various matrixes: Fibronectin (FN), Netrin-4, Collagen I (Col. I), and Poly-L-Lysin (PLL) at 10 μg/mL. (E) In vitro tube formation by HMVEC-dLys under different doses of Netrin-4, FGF-2 (bF), HGF, VEGF-A (VA), VEGF-C (VC), or complete media (CM). (F) Inhibition of serum deprivation–induced HMVEC-dLys apoptosis under different doses of Netrin-4, FGF-2, VEGF-A (VA), VEGF-C (VC), and complete media (CM). *P < .05.

Article Snippet: Cell culture and in vitro assays Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum.

Techniques: Migration, Control, Boyden Chamber Assay, In Vitro, Inhibition

Journal: Blood

Article Title: Netrin-4 induces lymphangiogenesis in vivo

doi: 10.1182/blood-2009-11-252338

Figure Lengend Snippet: Netrin-4 activates intracellular signaling pathways of HMVEC-dLys. Determination of the phosphorylation of Akt (Ser 473; Serine 473, panels A and D, respectively), p42/p44 (Thr 202/Tyr 204; Threonin 202/Tyrosine 204, panels B and E, respectively), and Ribosomal protein S6 (Ser 235/236; Serine 235/236, panels C and F) by Western blotting after Netrin-4 treatment of HMVEC-dLy. Experiments were performed in triplicate.

Article Snippet: Cell culture and in vitro assays Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: Blood

Article Title: Netrin-4 induces lymphangiogenesis in vivo

doi: 10.1182/blood-2009-11-252338

Figure Lengend Snippet: Netrin-4 induces in vitro lymphatic permeability. (A) Induction of GTP-RhoA and Rac1 by Netrin-4, VEGF-A, and VEGF-C treatment of HMVEC-dLys. (B) Stimulation of the phosphorylation of Tyrosine 416 of Src kinase family (SFK, Tyr416) and the Tyrosin 861 but not the Tyrosine 391 of focal adhesion kinase (FAK; Tyr861 and Tyr391) by Netrin-4 (500 ng/mL) and VEGF-C (500 ng/mL). (C) Measurement of the electrical resistance of the cell monolayer over 24 hours using the ECIS system or (D) immunostained using an anti–VE-cadherin antibody to visualize cell junctions (scale bar: 50 μm) of HMVEC-dLys seeded either on Fibronectin or Fibronectin plus Netrin-4. (E) Membrane fraction proteins prepared from HMVEC-dLys seeded as previously mentioned in panels C and D analyzed for ZO-1, VE-cadherin, and beta-catenin expression (equivalent loading assessed by coomassie blue staining). (F) Control, Netrin-4, VEGF-C overexpressing MCF7 tumor sections stained for the cell junction protein ZO-1 or the lymphatic marker LYVE1 (scale bar: 20 μm). Data presented in panels A through E are from 1 experiment and representative of 2 independent experiments. Pictures were taken on an Olympus IX71 microscope, at 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software.

Article Snippet: Cell culture and in vitro assays Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum.

Techniques: In Vitro, Permeability, Phospho-proteomics, Membrane, Expressing, Staining, Control, Marker, Microscopy, Software