Journal: International Journal of Oral Science

Article Title: Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-κB axis dependent manner

doi: 10.1038/s41368-020-00096-z

Figure Lengend Snippet: P. gingivalis infection damages vascular endothelial integrity. a The weight of mice in the control and P. gingivalis (Pg) groups at 6, 7, 8, 9, 10, 11, and 12 weeks. Representative images after haematoxylin-eosin staining (H&E) and CD31 immunohistochemistry (IHC) of brachiocephalic artery ( b ) and aortic arch ( c ) tissue from the Balb/c mice with Pg tail-vein injection for 6 weeks ( n = 5). Black arrow, the site where endothelial integrity was destroyed. Data were analysed using two-tailed unpaired t -tests ( a ) and are presented as the mean ± SD

Article Snippet: Human umbilical vein endothelial cells (HUVECs; ScienCell) and human aortic endothelial cells (HAECs; ScienCell) were cultured in endothelial cell medium (ScienCell) supplemented with 5% foetal bovine serum and 1% endothelial cell growth factors.

Techniques: Infection, Control, Staining, Immunohistochemistry, Injection, Two Tailed Test

Journal: International Journal of Oral Science

Article Title: Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-κB axis dependent manner

doi: 10.1038/s41368-020-00096-z

Figure Lengend Snippet: P. gingivalis inhibits proliferation and stimulates endothelial mesenchymal transformation (EndMT) in cultured endothelial cells. a The viability of human umbilical vein endothelial cells (HUVECs) treated with P. gingivalis (Pg) at different concentrations was measured by CCK-8 assays. MOI, multiplicity of infection. b EdU staining of HUVECs after Pg exposure for 48 h. Nuclei were stained with DAPI (blue). c The quantitative data of ( b ). d Immunofluorescence (IF) staining of Ki67 (green) in HUVECs with or without Pg infection for 48 h. e The quantitative data of ( d ). f EdU staining of human aortic endothelial cells (HAECs) after Pg exposure for 48 h. Nuclei were stained with DAPI (blue). g The quantitative data of ( f ). h HUVECs showed remarkable morphological changes after Pg treatment for 48 h. Representative images are presented. i Relative CD31, VE-cadherin (VE-cad), ACTA2 and FSP-1 mRNA levels were measured by real-time polymerase chain reaction (qRT-PCR) in the control or Pg-treated HUVECs. IF staining of CD31 ( j ) and α-SMA ( k ) in HUVECs after Pg infection or not. l Western blot analysis of the EndMT-related protein levels in the HUVECs after Pg exposure. m The quantitative data of l . n Western blot analysis of EndMT-related protein levels in HAECs after Pg exposure. o The quantitative data of ( n ). p Transwell assays were used to test the migration of the control and Pg-infected HUVECs. q The quantitative data of ( p ). Data were analysed using two-tailed unpaired t -tests ( c , e , g , i , m , o , q ) and are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the control group

Article Snippet: Human umbilical vein endothelial cells (HUVECs; ScienCell) and human aortic endothelial cells (HAECs; ScienCell) were cultured in endothelial cell medium (ScienCell) supplemented with 5% foetal bovine serum and 1% endothelial cell growth factors.

Techniques: Transformation Assay, Cell Culture, CCK-8 Assay, Infection, Staining, Immunofluorescence, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Control, Western Blot, Migration, Two Tailed Test

Journal: International Journal of Oral Science

Article Title: Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-κB axis dependent manner

doi: 10.1038/s41368-020-00096-z

Figure Lengend Snippet: P. gingivalis triggers endothelial apoptosis. a Apoptosis in vivo was assessed by TUNEL staining in brachiocephalic artery tissue from the Balb/c mice treated with or without P. gingivalis (Pg) ( n = 5). b Apoptosis was evaluated by flow cytometry of human umbilical vein endothelial cells (HUVECs) treated with Pg for 24 h. c The quantitative data of ( b ). d Western blot analysis of the indicated protein levels in HUVECs after Pg stimulation for 24 h. e The quantitative data of ( d ). f Western blot analysis of the indicated protein levels in human aortic endothelial cells (HAECs) after Pg stimulation for 24 h. g The quantitative data of ( f ). Data were analysed using a one-way ANOVA ( c ) or two-tailed unpaired t -tests ( e , g ) and are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the control group

Article Snippet: Human umbilical vein endothelial cells (HUVECs; ScienCell) and human aortic endothelial cells (HAECs; ScienCell) were cultured in endothelial cell medium (ScienCell) supplemented with 5% foetal bovine serum and 1% endothelial cell growth factors.

Techniques: In Vivo, TUNEL Assay, Staining, Flow Cytometry, Western Blot, Two Tailed Test, Control

Journal: International Journal of Oral Science

Article Title: Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-κB axis dependent manner

doi: 10.1038/s41368-020-00096-z

Figure Lengend Snippet: P. gingivalis damages the integrity of endothelial cells through TLRs. a Western blot analysis of TLR signalling-related protein levels in human umbilical vein endothelial cells (HUVECs) after P. gingivalis (Pg) exposure for 2 h or 24 h. b The quantitative data of ( a ). c Confocal images of IRAK1 expression in HUVECs treated with Pg and/or the TLR antagonist OxPAPC (30 μg·mL −1 ) for 2 h. d Western blot analysis of the p-IRAK1, IRAK1, and IRAK4 protein levels in the HUVECs treated with Pg and/or OxPAPC (10, 20, 30 μg·mL −1 ) for 2 h. e The quantitative data of ( d ). f The viability of HUVECs treated with Pg and/or OxPAPC at different timepoints was measured by CCK-8 assays. g EdU staining of HUVECs treated with Pg and/or OxPAPC (30 μg·mL −1 ) for 48 h. Nuclei were stained with DAPI (blue). h The quantitative data of ( g ). i HUVECs showed remarkable morphological changes after Pg and/or OxPAPC treatment for 48 h. Representative images are presented. j Western blot analysis of the EndMT-related protein levels in the HUVECs treated with Pg and/or OxPAPC (10, 20, 30 μg·mL −1 ) for 72 h. k Transwell assays were used to test the migration of HUVECs treated with Pg and/or OxPAPC (30 μg·mL −1 ) for 48 h. l The quantitative data of ( k ). m Apoptosis was evaluated by flow cytometry of the HUVECs treated with Pg and/or OxPAPC (30 μg·mL −1 ) for 24 h. n The quantitative data of ( m ). Data were analysed using a two-tailed unpaired t -test ( b ) or one-way ANOVA ( e , h , l , n ) or a two-way ANOVA ( f ) and are presented as the mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the control group. ns = not significant. # P < 0.05; ## P < 0.01; ### P < 0.001 compared to the Pg group

Article Snippet: Human umbilical vein endothelial cells (HUVECs; ScienCell) and human aortic endothelial cells (HAECs; ScienCell) were cultured in endothelial cell medium (ScienCell) supplemented with 5% foetal bovine serum and 1% endothelial cell growth factors.

Techniques: Western Blot, Expressing, CCK-8 Assay, Staining, Migration, Flow Cytometry, Two Tailed Test, Control

Journal: International Journal of Oral Science

Article Title: Porphyromonas gingivalis disrupts vascular endothelial homeostasis in a TLR-NF-κB axis dependent manner

doi: 10.1038/s41368-020-00096-z

Figure Lengend Snippet: NF-kappaB signalling plays a critical role in P. gingivalis -destroyed endothelial homeostasis. a Western blot analysis of NF-kappaB (NF-κB) signalling-related protein levels in human umbilical vein endothelial cells (HUVECs) after P. gingivalis (Pg) exposure for 2 h. b The quantitative data of ( a ). c Confocal images of p65 expression in the HUVECs treated with Pg and/or NF-κB inhibitor BAY 11-7082 (BAY, 1 μmol·L −1 ) for 2 h. d Western blot analysis of p-p65, p65, and IκBα protein levels in the HUVECs treated with Pg and/or BAY (0.1, 0.5, 1 μmol·L −1 ) for 2 h. e The quantitative data of ( d ). f EdU staining of the HUVECs treated with Pg and/or BAY (1 μmol·L −1 ) for 48 h. Nuclei were stained with DAPI (blue). g The quantitative data of ( f ). h Transwell assays were used to test the migration of HUVECs treated with Pg and/or BAY (1 μmol·L −1 ) for 48 h. i The quantitative data of ( h ). j Apoptosis was evaluated by flow cytometry of the HUVECs treated with Pg and/or BAY (1 μmol·L −1 ) for 24 h. k The quantitative data of ( j ). Data were analysed using a two-tailed unpaired t -test ( b ) or one-way ANOVA ( e , g , i , k ) and are presented as the means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001 compared to the control group. ns = not significant. # P < 0.05; ## P < 0.01; ### P < 0.001 compared to the Pg group

Article Snippet: Human umbilical vein endothelial cells (HUVECs; ScienCell) and human aortic endothelial cells (HAECs; ScienCell) were cultured in endothelial cell medium (ScienCell) supplemented with 5% foetal bovine serum and 1% endothelial cell growth factors.

Techniques: Western Blot, Expressing, Staining, Migration, Flow Cytometry, Two Tailed Test, Control

Journal: Journal of Cellular Physiology

Article Title: Osteocalcin does not influence acute or chronic inflammation in human vascular cells

doi: 10.1002/jcp.29231

Figure Lengend Snippet: Protein secretion responses in HAECs after acute inflammation. Mean % change relative to vehicle and SEM of secreted cell proteins VCAM‐1, ICAM‐1, IL‐6, endothelin, IL‐10, IL‐8, and MCP‐1 when treated with vehicle, ucOCN (10 ng/mL), an inflammatory protocol (IFN‐γ and TNF‐α, both 10 ng/ml, 8 hr followed by 16 hr, respectively), or inflammatory protocol and ucOCN. Data were normalized to total protein content. Data were analyzed by one‐way ANOVA. **** indicates p < .001 compared with vehicle. HAEC, human aortic endothelial cell; ICAM‐1, intracellular adhesion molecule‐1; IL, interleukin; inflm, inflammatory protocol; MCP‐1, monocyte chemoattractant protein‐1; ucOCN, uncarboxylated osteocalcin; SEM , standard error of mean; VCAM‐1, vascular cell adhesion molecule‐1

Article Snippet: OCN has previously been shown to increase nitric oxide production and prevent free fatty acid‐induced apoptosis in human aortic endothelial cells (HAECs); increase angiogenesis in a chick embryo in vivo model; increase proliferation in HAECs and HASMCs; and increase glucose metabolism and promote vascular calcification in OCN‐over expressing mouse chondrocytes and vascular smooth muscle cells (Cantatore, Crivellato, Nico, & Ribatti, ; Idelevich, Rais, & Monsonego‐Ornan, ; Jung et al., ; Millar, Anderson, & O'sullivan, ).

Techniques:

Journal: Journal of Cellular Physiology

Article Title: Osteocalcin does not influence acute or chronic inflammation in human vascular cells

doi: 10.1002/jcp.29231

Figure Lengend Snippet: Intracellular signaling responses in HAECs. Luminex® xMAP® technology was used to detect changes in phosphorylated CREB (pS133), JNK (pT183/pY185), NFkB (pS536), p38 (pT180/pY182), ERK (pT185/pY187), Akt (pS473), p70 S6K (pT412), STAT3 (pS727), and STAT5A/B (pY694/699; 48‐680MAG; Milliplex™; Merck Millipore) in cell lysates when treated with vehicle or ucOCN (10 ng/ml) with and without inflammatory stimulus (IFN‐γ and TNF‐α) for 24 hr. Data were analyzed by one‐way ANOVA with multiple comparisons to vehicle corrected for by Dunnett's test. Data are given as means with error bars representing SEM . *denotes a significant difference compared with vehicle (* p < .05. ** p < .01, *** P < .005, **** p < .001). HAEC, human aortic endothelial cell; IFN‐γ, interferon‐γ; inflm, inflammatory protocol (8 hr of IFN‐γ 10 ng/ml followed by addition of TNF‐α 10 ng/mL for 16 hr); SEM , standard error of mean; TNF‐α, tumor necrosis factor‐α; ucOCN, uncarboxylated osteocalcin

Article Snippet: OCN has previously been shown to increase nitric oxide production and prevent free fatty acid‐induced apoptosis in human aortic endothelial cells (HAECs); increase angiogenesis in a chick embryo in vivo model; increase proliferation in HAECs and HASMCs; and increase glucose metabolism and promote vascular calcification in OCN‐over expressing mouse chondrocytes and vascular smooth muscle cells (Cantatore, Crivellato, Nico, & Ribatti, ; Idelevich, Rais, & Monsonego‐Ornan, ; Jung et al., ; Millar, Anderson, & O'sullivan, ).

Techniques: Luminex

Journal: Journal of Cellular Physiology

Article Title: Osteocalcin does not influence acute or chronic inflammation in human vascular cells

doi: 10.1002/jcp.29231

Figure Lengend Snippet: IL‐6 secretion, LDH activity, IL‐10 secretion, and total protein content in HAECs after chronic inflammation with or without OCN. The effects of ucOCN on secretion of IL‐6 without inflammation (a) and with inflammation (b) after 0, 48, 96, and 144 hr. The effect of ucOCN on LDH activity was measured by NADH concentration without inflammation (c) and with inflammation (d) after 0, 48, 96, and 144 hr. IL‐10 secretion was measured after 144 hr (e). Total protein content was measured by BCA assay at the end of the experiment. ucOCN (10 ng/ml) was added every 48 hr. Total n = 9 from three experimental repeats. Data are given as means with error bars representing SEM . *denotes a significant difference compared with vehicle, analyzed by one‐way ANOVA (** p < .01, *** p < .005). ANOVA, analysis of variance; HAEC, human aortic endothelial cell; IL, interleukin; IFN‐γ, interferon‐γ; inflm, inflammatory protocol (8 hr of IFN‐γ followed by addition of TNF‐α; both 5 ng/ml); LDH, lactate dehydrogenase; NADH, nicotinamide adenine dinucleotide H; SEM , standard error of mean; TNF‐α, tumor necrosis factor‐α; ucOCN, uncarboxylated osteocalcin

Article Snippet: OCN has previously been shown to increase nitric oxide production and prevent free fatty acid‐induced apoptosis in human aortic endothelial cells (HAECs); increase angiogenesis in a chick embryo in vivo model; increase proliferation in HAECs and HASMCs; and increase glucose metabolism and promote vascular calcification in OCN‐over expressing mouse chondrocytes and vascular smooth muscle cells (Cantatore, Crivellato, Nico, & Ribatti, ; Idelevich, Rais, & Monsonego‐Ornan, ; Jung et al., ; Millar, Anderson, & O'sullivan, ).

Techniques: Activity Assay, Concentration Assay, BIA-KA

Journal: Cell Death & Disease

Article Title: Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis

doi: 10.1038/s41419-017-0257-3

Figure Lengend Snippet: a – b The protein levels of Caspase-1, IL-18, and IL-1β were upregulated in HAECs after treatment with nicotine for 24 h, as indicated by western blot results. GAPDH was used as an internal control. c The relative mRNA levels of pro-inflammatory cytokines (IL-1β, IL-18) and Caspase-1 were upregulated in HAECs after treatment with nicotine for 24 h. * P < 0.05, ** P < 0.01. The data are represented as mean ± SEM ( n = 3–5). d Caspase-1 (green) and TUNEL (red) double-positive cells were increased in the presence of nicotine. The nuclei were stained blue with DAPI. Magnification: ×200. Scale bar = 100 μm. e The relative LDH release was elevated in nicotine-treated endothelial cells ( n = 3). ** P < 0.01. f The percentage of PI (red) positive cells were increased in HAECs after treatment with nicotine (left: the representative photographs, right: the quantification of PI positive cells). Magnification: ×200. Scale bar = 500 μm. ** P < 0.01, *** P < 0.001. The data are represented as mean ± SEM ( n = 4)

Article Snippet: Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Western Blot, Control, TUNEL Assay, Staining

Journal: Cell Death & Disease

Article Title: Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis

doi: 10.1038/s41419-017-0257-3

Figure Lengend Snippet: HAECs were pretreated with caspase-1 inhibitor (VX-765, 10 μM) for 1 h, and then the cells were incubated with nicotine (1 μM) for 24 h. a VX-765 inhibited the protein expression of pro-caspase-1, caspase-1, IL-18, and IL-1β in nicotine-treated endothelial cells. b – e Quantitative analysis of pyroptosis-associated protein expression. GAPDH was used as an internal control ( n = 4). f Pyroptotic cell death was determined by LDH release, and the relative LDH activity was inhibited by VX-765 ( n = 5). g Double staining of PI (red) and Hoechst 33342 (blue) (left: the representative photographs, right: the quantification of PI positive cells). The increased percentage of PI positive cells in nicotine treated endothelial cells were reduced after pretreatment with VX-765. The data are represented as mean ± SEM ( n = 4). Magnification: ×200. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Incubation, Expressing, Control, Activity Assay, Double Staining

Journal: Cell Death & Disease

Article Title: Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis

doi: 10.1038/s41419-017-0257-3

Figure Lengend Snippet: a The expression levels of NLRP3 and ASC were increased in HAECs treated with nicotine (0.1, 1 μM) for 24 h, as determined by western blot results. b The mRNA levels of NLRP3 and ASC were increased in HAECs treated with nicotine (0.1, 1 μM) for 24 h. c – d NLRP3 siRNA silenced the expression of NLRP3 at both protein and mRNA level. e – f The protein levels of Caspase-1, IL-1β, and IL-18 in nicotine-treated HAECs were inhibited after transfection with siNLRP3. NC indicates negative control ( n = 4). g The mRNA levels of caspase-1, IL-1β, and IL-18 were inhibited by siNLRP3 in the presence of nicotine ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001. h TUNEL (red) and caspase-1 (green) double-positive cells were decreased in nicotine-treated endothelial cell that transfected with siNLRP3. The nuclei were stained blue with DAPI. Magnification: ×200. Scale bar denotes 100 μm. i Pyroptotic cell death was determined by LDH release, and the relative LDH activity was suppressed by siNLRP3 in the presence of nicotine ( n = 3). * P < 0.05. j The percentage of PI positive cells was declined after transfection with siNLRP3 in nicotine-treated HAECs (left: the representative photographs, right: the quantification of PI positive cells). Magnification: ×200. Scale bar = 500 μm. ** P < 0.01. The data are represented as mean ± SEM ( n = 4)

Article Snippet: Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Transfection, Negative Control, TUNEL Assay, Staining, Activity Assay

Journal: Cell Death & Disease

Article Title: Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis

doi: 10.1038/s41419-017-0257-3

Figure Lengend Snippet: a Verification of silencing efficiency of ASC by siRNA in nicotine-treated HAECs. b The protein levels of Caspase-1, IL-1β, and IL-18 in nicotine-treated HAECs were decreased after transfection of siASC. NC indicates negative control. c The mRNA levels of Caspase-1, IL-1β, and IL-18 in nicotine-treated HAECs were decreased after transfection of siASC. ( n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001. d TUNEL (red) and caspase-1 (green) double-positive cells were reduced by siASC in the presence of nicotine. Magnification: ×200. Scale bar denotes 100 μm. e Pyroptotic cell death was determined by LDH release, and the relative LDH activity was suppressed by ASC siRNA ( n = 3). ** P < 0.01. f The percentage of PI positive cells was declined after transfection with siASC in nicotine-treated HAECs. (left: the representative photographs, right: the quantification of PI positive cells). Magnification: ×200. Scale bar = 500 μm. ** P < 0.01. The data are represented as mean ± SEM ( n = 4)

Article Snippet: Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Transfection, Negative Control, TUNEL Assay, Activity Assay

Journal: Cell Death & Disease

Article Title: Nicotine promotes atherosclerosis via ROS-NLRP3-mediated endothelial cell pyroptosis

doi: 10.1038/s41419-017-0257-3

Figure Lengend Snippet: a Nicotine increased intracellular ROS level of HAECs, and this increase was inhibited by N-acetyl-cysteine (NAC, 5 mM). b Nicotine-induced the upregulation of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 mRNA levels were decreased by NAC ( n = 3–5). c – d NAC pretreatment suppressed the upregulation of Caspase-1, IL-1β, and IL-18 protein levels in the presence of nicotine. * P < 0.05, ** P < 0.01, *** P < 0.001. e TUNEL (red) and Caspase-1 (green) double-positive cells were decreased when pretreatment with NAC. Scale bar indicates 100 μm. Magnification: ×200. f Pyroptotic cell death was determined by LDH release, and the relative LDH activity was decreased when pretreated with NAC ( n = 5). * P < 0.05, ** P < 0.01

Article Snippet: Human aortic endothelial cells (HAECs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: TUNEL Assay, Activity Assay