Journal: The FEBS journal

Article Title: Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells.

doi: 10.1111/febs.17349

Figure Lengend Snippet: Fig. 1. Pg OMVs increase vascular permeability in vivo and in vitro. (A) Representative dorsal skin images of mice in the Miles assay. Porphyromonas gingivalis (Pg) outer membrane vesicles (OMVs) containing 15 lg of protein (right side of the skin) or saline (left side of the skin) were singly injected into dorsal skin of BALB/ cAJc1 mice. Subsequently, 100 lL of 0.5% Evans blue was injected into the tail vein. After 10 min, the gross Evans blue dye level was visually observed on the skin surface in Pg OMVs- injected area (red circles) and saline-injected area (black circles). Four mice were used per group for each experiment and a typical image of visual discoloration is shown. (B) Human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were grown to confluence on the insert of a transwell system and treated with (n = 5) or without (none, n = 4) Pg OMVs (500 ngmL1) for 60 min. FITC-labeled dextran (1 mgmL1) was then added to the insert. After 10 min, the FITC-labeled dextran leakage from the apical compartment of the insert to the bottom well was quantified by measuring the fluorescence intensity. Student’s t-test was used for statistical analysis. Error bars represent standard deviation. **P < 0.01 compared with no-treated cells (None).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMECs) (C12281; PromoCell GmbH, Heidelberg, Germany) were purchased from PromoCell and maintained in MV medium prepared using an Endothelial Cell Growth Medium MV Kit (C-22121; PromoCell).

Techniques: Permeability, In Vivo, In Vitro, Membrane, Saline, Injection, Labeling, Standard Deviation

Journal: The FEBS journal

Article Title: Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells.

doi: 10.1111/febs.17349

Figure Lengend Snippet: Fig. 2. Pg OMVs induce stress fiber formation in HUVECs and HPMECs. HUVECs (A, B) and HPMECs (C, D) were cultured in monolayers and treated with (B, D, F) or without (none; A, C, E) 500 ngmL1 of Porphyromonas gingivalis (Pg) outer membrane vesicles (OMVs) for 60 min. Cells were fixed and stained with vascular endothelial-cadherin (VEc) to label adhesion junctions (AJs) (magenta) (A, B). F-actin was labeled with FITC-phalloidin (green) (C, D), and the nuclei were stained with Hoechst 33342 (white) and merged (E, F). High-magnification merged images (2.59) are shown in (B) and (D). Arrows indicate radical stress fibers. The arrowhead represents focal AJs connected to stress fibers. Scale bars indicate 20 lm. n = 4 in each group. Four samples were set up in each group (n = 4), and the typical photographs were shown from four independent experiments. (E). F-actin was labeled with FITC-phalloidin (green) in HUVECS and HPMECs treated with or without 500 ngmL1 of Pg OMVs for 60 min (n = 4 in each group), and green fluorescence intensity were measured. Student’s t-test was used for statistical analysis. Error bars represent standard deviation. **P < 0.01 compared with no-treated cells (None).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMECs) (C12281; PromoCell GmbH, Heidelberg, Germany) were purchased from PromoCell and maintained in MV medium prepared using an Endothelial Cell Growth Medium MV Kit (C-22121; PromoCell).

Techniques: Cell Culture, Membrane, Staining, Labeling, Standard Deviation

Journal: The FEBS journal

Article Title: Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells.

doi: 10.1111/febs.17349

Figure Lengend Snippet: Fig. 3. Pg OMVs downregulate the level of VEc protein but not mRNA in HUVECs and HPMECs. (A) Human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were treated with 500 ngmL1 Porphyromonas gingivalis (Pg) outer membrane vesicles (OMVs). Subsequently, vascular endothelial-cadherin (VEc) expression was analyzed via western blotting at indicated periods. (B) HUVECs and HPMECs were treated with Pg OMVs for 0 or 180 min, and 60 min, respectively. (A) The cell lysates were analyzed via western blotting using antibodies for VEc and GAPDH. (B) Densitometric analysis of each band was performed using IMAGEJ, and the VEc/GAPDH ratio in each group were presented and analyzed using Student’s t-test (n = 4 in each group). Values are shown as the fold change in expression levels compared to that in the 0 min group. Error bars represent standard deviation. *P < 0.05, **P < 0.01 compared with 0 min. (C) HUVECs were treated with 500 ngmL1 Pg OMVs for 0–180 min (n = 4). (D) HUVECs were treated with 0–500 ngmL1 Pg OMVs for 180 min (n = 4). VEc mRNA expression was analyzed using real-time PCR. Values are shown as the fold change in expression levels compared to that in the 0 min group. Student’s t-test was used for statistical analysis (n = 4 in each group; n.s., not significant). Error bars represent standard deviation.

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMECs) (C12281; PromoCell GmbH, Heidelberg, Germany) were purchased from PromoCell and maintained in MV medium prepared using an Endothelial Cell Growth Medium MV Kit (C-22121; PromoCell).

Techniques: Membrane, Expressing, Western Blot, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: The FEBS journal

Article Title: Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells.

doi: 10.1111/febs.17349

Figure Lengend Snippet: Fig. 4. Pg OMVs accelerate VEc degradation through the lysosomal pathway in HUVECs. (A) Human umbilical vein endothelial cells (HUVECs) were pretreated with (+) (B, C, E, F) or without () (A, D) chloroquine for 30 min. Subsequently, cells were treated with (D–F) or without (A–C) 500 ngmL1 Porphyromonas gingivalis (Pg) outer membrane vesicles (OMVs) for 180 min before fixation and subjected to vascular endothelial-cadherin (VEc) staining for immunofluorescence analysis. Nuclei were stained with Hoechst 33342. High-magnification images (49) of (B) and (E) are shown (C, F). Scale bar, 20 lm. Arrowheads indicate the accumulation of intracellular vesicular VEc. Four samples were set up in each group (n = 4), and the typical photographs were shown from three independent experiments. (B) Vesicular VEc was induced by same method shown as (A). VEc and lysosome-associated membrane protein 1 (LAMP-1) were co-stained with specific antibodies for VEc (A, E) or LAMP-1 (B, F). Nuclei were stained with Hoechst 33342 and the microscopic images of the same field were merged (C, G). High- magnification images (49) of the merge are shown (D, H). Scale bars indicate 20 lm. Arrowheads indicate vesicular VEc surrounded by LAMP- 1. (I) The merged white fluorescent intensity and green fluorescent intensity around nucleus were quantified. The ratio of merged (white)/ LAMP-1 (green) is shown as the co-localization coefficients of VEc and LAMP-1. Student’s t-test was used for statistical analysis (n = 4, samples; n = 8, field of view, in each group). Error bars represent standard deviation. **P < 0.01 compared with no-treated cells (None). (C) The same samples used in (B) were subjected to confocal microscopy (A, C), and high-magnification images (49) are shown in (B, D). VEc and LAMP-1 double-staining revealed co-localization in some vesicles induced by Pg OMVs (D, arrows).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMECs) (C12281; PromoCell GmbH, Heidelberg, Germany) were purchased from PromoCell and maintained in MV medium prepared using an Endothelial Cell Growth Medium MV Kit (C-22121; PromoCell).

Techniques: Membrane, Staining, Standard Deviation, Confocal Microscopy, Double Staining

Journal: The FEBS journal

Article Title: Porphyromonas gingivalis outer membrane vesicles increase vascular permeability by inducing stress fiber formation and degrading vascular endothelial-cadherin in endothelial cells.

doi: 10.1111/febs.17349

Figure Lengend Snippet: Fig. 6. Graphical summary. In healthy individuals, the cell–cell junction is constructed with linear adhesion junctions (AJs) supported by circumferential actin bundles. Therefore, the endothelial barrier was strong and had normal vascular permeability. Porphyromonas gingivalis (Pg) outer membrane vesicles (OMVs) released during periodontal disease may induce Rho activation. Activated Rho kinases increase stress fiber formation, thereby weakening the endothelial barrier by forming focal AJs. Pg OMVs-activated Rho kinases also promote vascular endothelial-cadherin (VEc) internalization via endocytosis and subsequent degradation via the endosomal and lysosomal pathways, leading to a decrease in VEc levels.

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMECs) (C12281; PromoCell GmbH, Heidelberg, Germany) were purchased from PromoCell and maintained in MV medium prepared using an Endothelial Cell Growth Medium MV Kit (C-22121; PromoCell).

Techniques: Construct, Permeability, Membrane, Activation Assay

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Substrate stiffness influences BM‐MSC EV production and bioactivity. (a) EV production as quantified by EVs per cell from BM‐MSCs seeded on Sylgard 184 PDMS substrates with different base‐to‐crosslinker ratios. EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted ( n = 3). (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in EV treatments or growth or basal endothelial media, seeded in Matrigel‐coated wells, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. All data are representative of at least three independent experiments ( n = 3). Statistical significance was determined by ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: To measure in vitro angiogenesis, 48‐well plates were coated with 60 μL of growth factor reduced Matrigel (Corning; 356230) and incubated at 37°C for 30 min. P4 HUVECs were then seeded at 35,000 cells/well with either endothelial growth media (PromoCell; C‐22121) with 1% penicillin–streptomycin (positive control), endothelial basal media (negative control), or endothelial basal media (PromoCell; C‐22221) with 0.1% FBS and 1% penicillin–streptomycin with 5E9 EVs/mL.

Techniques: Isolation, Microscopy

Journal: Bioengineering & Translational Medicine

Article Title: Mesenchymal stem cell extracellular vesicle vascularization bioactivity and production yield are responsive to cell culture substrate stiffness

doi: 10.1002/btm2.10743

Figure Lengend Snippet: Softer 184:527 PDMS substrates improve the angiogenic bioactivity of BM‐MSC EVs. (a) EV production quantified as EV per cell from BM‐MSCs seeded on each substrate made with different ratios of Sylgard 184 and Sylgard 527 ( n = 2). EVs used for this data were from 1 day of collection and isolated and counted separately from the conditioned media from the other 2 days. After media collection, cells were trypsinized and counted. (b) After a scratch was induced, HUVECs were treated with BM‐MSC EVs from the different substrates or growth or basal media, and percent gap closure after 20 h was evaluated via microscopy ( n = 3). (c) HUVECs were resuspended in the different EV treatments or growth or basal endothelial basal media, and tube formation after 3–6 h was quantified by the number of loops that had formed ( n = 3). All values expressed as mean ± SD. Statistical significance was determined by ANOVA; * p < 0.05, ** p < 0.01, and **** p < 0.0001.

Article Snippet: To measure in vitro angiogenesis, 48‐well plates were coated with 60 μL of growth factor reduced Matrigel (Corning; 356230) and incubated at 37°C for 30 min. P4 HUVECs were then seeded at 35,000 cells/well with either endothelial growth media (PromoCell; C‐22121) with 1% penicillin–streptomycin (positive control), endothelial basal media (negative control), or endothelial basal media (PromoCell; C‐22221) with 0.1% FBS and 1% penicillin–streptomycin with 5E9 EVs/mL.

Techniques: Isolation, Microscopy