Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 1 Metformin promotes angiogenesis under hypoxic conditions in vitro. Representative images (a) of the transwell migration assay with quantification of crystal violet-stained migrated HMECs (b) treated with metformin under different oxygen concentrations (21% and 1% O2). Met: metformin. Scale bar: 100 μm. n = 3 per group. Representative images (c) of tube formation of HMECs on Matrigel with quantification of the total branching points (d), total tube length (e), and total loop numbers (f). Scale bar: 200 μm. n = 3 per group. g CCK-8 analysis of the proliferation of HMECs. n = 4 per group. h qRT‒PCR analysis of the mRNA levels of the HIF-1α target genes Vegfa and Lrg1 in HMECs with or without metformin treatment under different oxygen concentrations. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques: In Vitro, Transwell Migration Assay, Staining, CCK-8 Assay

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 3 Metformin promotes type H vessel formation in osteoporotic fracture mice. a Representative CD31 and Emcn coimmunostaining images (left) with quantification of the type H vessel ratio in calluses (right) from the osteoporotic mice treated with PBS, Met, PTH, and ALN at 3, 6, and 9 weeks post-fracture. ca: callus. The dotted line represents the boundary of the callus. Met metformin, ALN alendronate, PTH parathyroid hormone. Scale bar: 100 μm. n = 5 per group. b Representative Ki67 and Emcn coimmunostaining images (left) with quantification of the number of Ki67-positive endothelial cells in calluses (right) from the osteoporotic mice treated with PBS, Met, PTH, and ALN at 3, 6, and 9 weeks post-fracture. Scale bar: 100 μm. n = 5 per group. ELISAs for the serum (c) and bone marrow (d) concentrations of VEGFA at 9 weeks post-osteoporotic fracture. n = 8 per group; Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques:

Journal: Bone research

Article Title: Metformin accelerates bone fracture healing by promoting type H vessel formation through inhibition of YAP1/TAZ expression.

doi: 10.1038/s41413-023-00279-4

Figure Lengend Snippet: Fig. 4 Metformin promotes the expression of HIF-1α by inhibiting the expression of YAP1/TAZ in HMECs under hypoxic conditions. a qRT‒PCR analysis of the inhibitory efficiency of siRNAs targeting HIF-1α. n = 3 per group. b qRT‒PCR analysis of the expression of HIF-1α and its target genes Vegfa and Lrg1 in the si-HIF-1α-transfected HMECs with or without metformin treatment under hypoxic conditions (1% O2). Met: metformin. n = 3 per group. Immunofluorescence staining images and quantification showing the protein levels of HIF-1α (c), YAP1 (d), and TAZ (e) in the HMECs treated with PBS (control) or metformin under hypoxic conditions. Scale bar: 20 μm. n = 9 per group. f qRT‒PCR analysis of the inhibitory efficiency of siRNAs targeting YAP1 or TAZ. n = 3 per group. g Immunofluorescence staining images and quantification showing the protein level of HIF-1α in the hypoxia-cultured HMECs from the si-Con, si-YAP1, si-TAZ, si-Y/T, and si-Y/T + Met groups. Y/T: YAP1 and TAZ. Scale bar: 20 μm. n = 3 per group. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) The concentrations of OCN and VEGFA were determined using the Mouse OCN (Elabscience, E-EL-M0864c, Wuhan, China) or VEGFA (Elabscience, E-EL-M1292c) ELISA kit according to the manufacturers’ instructions.

Techniques: Expressing, Transfection, Staining, Control, Cell Culture

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: 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 μg 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 μL 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 ng·mL −1 ) for 60 min. FITC‐labeled dextran (1 mg·mL −1 ) 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) (C‐12281; 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, Fluorescence, 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: 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 ng·mL −1 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 (2.5×) are shown in (B) and (D). Arrows indicate radical stress fibers. The arrowhead represents focal AJs connected to stress fibers. Scale bars indicate 20 μm. 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 ng·mL −1 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) (C‐12281; 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, Fluorescence, 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: 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 ng·mL −1 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 ng·mL −1 Pg OMVs for 0–180 min ( n = 4). (D) HUVECs were treated with 0–500 ng·mL −1 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) (C‐12281; 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: 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 ng·mL −1 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 (4×) of (B) and (E) are shown (C, F). Scale bar, 20 μm. 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 (4×) of the merge are shown (D, H). Scale bars indicate 20 μm. 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 (4×) 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) (C‐12281; 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, Immunofluorescence, 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: Rho kinases are involved in Pg OMVs‐induced permeability in HUVECs. (A) The hyperpermeability signaling pathway promoted by actin filament rearrangement. (B) Human umbilical vein endothelial cells (HUVECs) were treated with 500 ng·mL −1 Porphyromonas gingivalis ( Pg ) outer membrane vesicles (OMVs) for the indicated periods and cofilin phosphorylation was assessed via western blotting. The immunoblots are representative figures of three independent experiments. (C) HUVECs were pretreated with 10 μ m Y27632 for 30 min before Pg OMVs challenge for 60 min (C, F, I). Cells were treated, fixed, and stained as described in Fig. . Arrows indicate the radical stress fibers (E, H). The arrowhead indicates focal adhesion junctions (AJs) connected to stress fibers (E, H). Bars indicate 20 μm. The fluorescence intensity of F‐actin labeled with FITC‐phalloidin (green, in D, E and F) was quantified (J). Student's t ‐test was used for statistical analysis ( n = 4 in each group). Error bars represent standard deviation. ** P < 0.01 compared with no‐treated cells (None). (D) HUVECs were pretreated with 10 μ m Y27632 for 30 min before Pg OMVs challenge for 180 min. Vascular endothelial‐cadherin (VEc) protein levels were analyzed via western blotting. WT, wild‐type; KDP136, gingipain deficient. (E) HUVECs were grown on Transwell inserts and pretreated with 10 μ m Y27632 for 30 min before Pg OMVs challenge for 60 min. Vascular permeability was assessed as shown in Fig. . Student's t ‐test was used for statistical analysis ( n = 4). Error bars represent standard deviation. ** P < 0.01.

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMECs) (C‐12281; 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, Membrane, Phospho-proteomics, Western Blot, Staining, Fluorescence, 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: 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) (C‐12281; 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