Journal: International Journal of Molecular Sciences

Article Title: Hypercholesterolemia-Induced HDL Dysfunction Can Be Reversed: The Impact of Diet and Statin Treatment in a Preclinical Animal Model

doi: 10.3390/ijms23158596

Figure Lengend Snippet: Effects of diet and rosuvastatin intervention on HDL antioxidant capacity. Changes in HDL antioxidant capacity are presented as percent antioxidant index (( A ), % AOI; measure for capacity to reverse LDL oxidation) and levels of conjugated dienes (( B – E ) product of lipid oxidation). Data are shown as mean ± SEM (( A ); for visual clarity of the graph) or SD (whiskers) over the period of 40 days. ( A ) includes all four groups, whereas ( B – E ) represent each group individually. Shapiro–Wilk test confirmed normality (alpha = 0.05), and data were analyzed by one-way ANOVA with Tukey’s multicomparison test and considered significant with a p -value < 0.05. For ( A ): * p < 0.05 in comparison to day 0; † p < 0.05 in comparison to day 10; # p < 0.05 compared to HC; § p < 0.05 in comparison to NC. For ( B – E ): * p < 0.05, ** p < 0.0001 in comparison to day 0; † p < 0.05 in comparison to day 10. d0–40: day 0–40; HC: hypercholesterolemic diet; NC: normocholesterolemic diet; +R/+rosu: + rosuvastatin; LDL: low-density lipoprotein.

Article Snippet: HDL AOI is based on the capacity of HDL particles to actively reverse LDL oxidation using 2,7-dichlorofluoresceindiacetate (H2DCFDA) (Invitrogen Inc., Carlsbad, CA, USA) and copper-oxidized LDL particles (Kalen Biomedical Inc., Savage, MD, USA).

Techniques:

Journal: Physiological Reports

Article Title: TRPV 4 is a regulator in P. gingivalis lipopolysaccharide‐induced exacerbation of macrophage foam cell formation

doi: 10.14814/phy2.14069

Figure Lengend Snippet: Pg LPS ‐triggered exacerbation of oxidized LDL ‐mediated macrophage foam cell formation is dependent on TRPV 4. (A) WT and TRPV 4 KO ( TRPV 4−/−) primary resident macrophages ( MRM s) were cultured for 48 h on collagen‐coated (10 μ g/mL) glass coverslips with or without Pg LPS (500 ng/mL), and then were incubated for an additional 20 h in the presence of native low‐density lipoproteins ( nLDL , 50 μ g/mL) or oxidized LDL (ox LDL , 50 μ g/mL). MRM s were stained with Oil‐Red‐O to examine macrophage foam cell formation. Representative images from five different fields per condition are shown (original magnification, 40×). (B) Bar graph shows quantitation of results in Figure A. Results are expressed as mean ± SEM . * P < 0.05 for ox LDL ‐treated WT cells with Pg LPS versus ox LDL ‐treated WT cells without Pg LPS , ** P < 0.01 for ox LDL ‐treated WT cells versus ox LDL ‐treated TRPV 4‐/‐ cells, and *** P < 0.001 for ox LDL treated TRPV 4−/− versus WT cells in presence of Pg LPS ; n = 500 cells/condition, Student's t ‐test. ns, not significant. (C) MRM s were cultured for 48 h with or without Pg LPS as above, and were then pretreated with or without TRPV 4 antagonist, GSK 219 (5 μ mol/L) and ox LDL or nLDL (50 μ g/mL) for 24 h. Cells were stained with Oil‐Red‐O to examine foam cell generation. Representative images from five different fields per condition are presented (original magnification, 40×). (D) Quantitation of results from Figure C. * P < 0.05 for ox LDL ‐treated cells with Pg LPS versus ox LDL ‐treated cells without Pg LPS , ** P < 0.01 for ox LDL ‐treated cells versus ox LDL ‐treated cells with GSK 219, and ## P < 0.001 for ox LDL plus Pg LPS ‐treated cells versus cells with GSK 219; n = 500 cells/condition, Student's t ‐test. (E) qRT ‐ PCR analysis was performed to determine levels of TNF ‐ α , IL ‐6, and IL ‐1 β mRNA in WT and TRPV 4 KO MRM s in the presence of nLDL or ox LDL using a TaqMan gene Expression Assay. All Ct values were normalized to gapdh levels. The experiment was repeated three times in quadruplicate. Student's t ‐test, * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: FLIPR Calcium 6 assay kit was purchased from Molecular Devices (Sunnyvale, CA) and macrophage colony‐stimulating factor (M‐CSF) was obtained from R & D. We prepared copper‐oxidized LDL (oxLDL) by incubating human native LDL (Stemcell Technologies; Vancouver, BC, Canada) with CuSO4 (5 μ m) for 12 h at 37°C, as described previously (Rahaman et al. , ).

Techniques: Cell Culture, Incubation, Staining, Quantitation Assay, Quantitative RT-PCR, Expressing

Journal: Physiological Reports

Article Title: TRPV 4 is a regulator in P. gingivalis lipopolysaccharide‐induced exacerbation of macrophage foam cell formation

doi: 10.14814/phy2.14069

Figure Lengend Snippet: TRPV 4 deficiency prevents Pg LPS ‐induced exacerbation of foam cell formation in response to increased matrix stiffness. (A) WT and TRPV 4−/− MRM s were grown on collagen‐coated (10 μ g/mL) soft (0.5 kP a) and stiff (8 kP a) polyacrylamide gels for 24 h with or without Pg LPS (500 ng/mL), followed by incubation with ox LDL (50 μ g/mL) for 24 h. Oil‐Red‐O staining was performed to examine foam cell generation. Representative images from five different fields per condition are shown (original magnification, 40×). (B) Bar graph shows quantitation of results in Figure A. Results are expressed as mean ± SEM . ** P < 0.01 for TRPV 4−/− cells on 8 kP a versus WT on 8 kP a; *** P < 0.001 for TRPV 4−/− on 8 kP a with Pg LPS versus WT cells on 8 kP a with Pg LPS ; ## P < 0.01 for WT cells on 8 kP a versus WT cells on 8 kP a with Pg LPS ; n = 50 cells/condition, Student's t ‐test.

Article Snippet: FLIPR Calcium 6 assay kit was purchased from Molecular Devices (Sunnyvale, CA) and macrophage colony‐stimulating factor (M‐CSF) was obtained from R & D. We prepared copper‐oxidized LDL (oxLDL) by incubating human native LDL (Stemcell Technologies; Vancouver, BC, Canada) with CuSO4 (5 μ m) for 12 h at 37°C, as described previously (Rahaman et al. , ).

Techniques: Incubation, Staining, Quantitation Assay

Journal: Physiological Reports

Article Title: TRPV 4 is a regulator in P. gingivalis lipopolysaccharide‐induced exacerbation of macrophage foam cell formation

doi: 10.14814/phy2.14069

Figure Lengend Snippet: TRPV 4 is required for ox LDL uptake but not its cell surface binding to macrophages. (A). WT and TRPV 4 KO MRM s were incubated with or without DiI‐labeled ox LDL (2.5 μ g/ mL ) for 60 min at 4°C to assess ox LDL binding. Representative images of DiI‐ox LDL binding on the macrophage surface are shown ( n = 5 fields/condition). (B) Quantification of results in Figure A using NIH ImageJ software. (C) WT and TRPV 4 KO MRM s were incubated with or without DiI‐labeled ox LDL (5 μ g/mL) for 30 min at 37°C, and ox LDL uptake was assessed. Representative images from five different fields per condition are shown (original magnification, 40×). DiI‐ox LDL uptake indicated by red fluorescence. (D) Bar graphs show mean DiI fluorescence intensity (mean ± SEM ) ( NIH ImageJ software). ** P < 0.01 for WT cells versus TRPV 4−/− without Pg LPS ; ## P < 0.01 for WT with Pg LPS versus WT without Pg LPS ; *** P < 0.001 for WT with Pg LPS versus TRPV 4−/− with Pg LPS ; n = 20 cells/condition.

Article Snippet: FLIPR Calcium 6 assay kit was purchased from Molecular Devices (Sunnyvale, CA) and macrophage colony‐stimulating factor (M‐CSF) was obtained from R & D. We prepared copper‐oxidized LDL (oxLDL) by incubating human native LDL (Stemcell Technologies; Vancouver, BC, Canada) with CuSO4 (5 μ m) for 12 h at 37°C, as described previously (Rahaman et al. , ).

Techniques: Binding Assay, Incubation, Labeling, Software, Fluorescence