Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: M1 macrophages express more PLXND1 than M2 macrophages in bifurcation lesions of ApoE −/− mice after 10 weeks and 20 weeks of high fat diet. (A) Adjacent sections of carotid bifurcation lesions were used to examine M1 and M2 macrophages by immunofluorescence staining separately. Immunofluorescence staining was performed with iNOS (green) or ARG1 (green), F4/80 (red), PLXND1 (pink), and DAPI (blue). Representative images are shown on the top (scale bar, 100 μm) and enlarged images of inserts are shown below (scale bar, 20 μm). (B) Intima PLXND1 + iNOS + F4/80 + M1 macrophages and PLXND1 + ARG1 + F4/80 + M2 macrophages were presented as number each slice. PLXND1 was expressed more in M1 macrophages rather than M2 macrophages. n = 4. ** P < 0.01, paired two-tailed t tests. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 expression level and location in process of atherosclerosis at bifurcations are evaluated by three-dimensional reconstruction. (A) 3D projections of PLXND1 (green) and iNOS (red) with autofluorescence at carotid bifurcations were depicted during atherosclerosis development by tissue clearing and light-sheet imaging. Representative images are shown (scale bar, 100 μm). Enlarged images of inserts are shown at right (scale bar, 10 μm). (B) Quantitative analyses of PLXND1 fluorescence of plaques show PLXND1 expression level increased by high-fat diet over time ( n = 4 mice; two carotid arteries/mice). (C) Quantitative analyses of iNOS fluorescence of plaques show iNOS expression level increased by high fat diet over time ( n = 4 mice; two carotid arteries/mice). (D) Co-localization of PLXND1 with iNOS in plaques at bifurcations was quantitatively analyzed ( n = 4 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. * P < 0.05, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Expressing, Imaging, Fluorescence

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Carotid bifurcation lesions are detected by probes targeting PLXND1. (A) Fluorescence imaging of isolated arteries from ApoE −/− mice were performed after intravenous injection of A12-Cy5, the molecular probes specific for PLXND1. A12-Cy5 showed stronger signals in carotid bifurcation lesions than common carotid arteries. (B) Arteries were conducted tissue clearing and light-sheet imaging. The images show that fluorescent probes accumulate inside the plaques (scale bar, 1 mm). (C) The ratio of fluorescent intensity between carotid bifurcation and common carotid artery were quantitatively analyzed at HFD 0w, 10w, 20w ( n = 3 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet.

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Fluorescence, Imaging, Isolation, Injection

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates LPS/IFN-γ-induced M1 macrophages polarization. (A) THP-1 cells were differentiated into macrophages, and then polarized to M1 or M2 macrophages by LPS/IFN-γ or IL-‍4/IL-13. Representative Western blots of PLXND1, iNOS (M1 marker), CD206 (M2 marker), CD163 (M2 marker), ARG1 (M2 marker) and GAPDH in M0, M1 and M2 type macrophages are shown. (B) Representative Western blots of PLXND1, p-STAT1/STAT1, p-STAT6/STAT6 and GAPDH in M0, M1 and M2 type macrophages are shown. (C) Quantitative analysis of PLXND1 protein expression demonstrates the highest level of PLXND1 protein in M1 macrophage. (D) THP-1 cells were transfected with shRNA to knockdown PLXND1. The knockdown efficiency was verified by western blotting (WB) and quantification of WB in (E). (F) THP-1 cells were transfected with sh-Control or sh-PLXND1 and further differentiated into macrophages via PMA. THP-1-derived macrophages were then incubated with or without LPS/IFN-γ. Representative Western blots of PLXND1, iNOS, and GAPDH are shown. (G) iNOS protein expression was quantitatively analyzed. Knockdown of PLXND1 attenuates the induction of iNOS, the M1 polarization marker in macrophages treated with LPS/IFN-γ. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Bonferroni's post-hoc test or unpaired two-tailed Student's t tests.

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Western Blot, Marker, Expressing, Transfection, shRNA, Knockdown, Control, Derivative Assay, Incubation, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates M1 macrophages polarization induced by oscillatory shear stress. (A) A schematic drawing of the experiment is outlined. THP-1-derived macrophages were treated with oxLDL and co-cultured with HUVECs exposed to laminar shear or oscillatory shear for 24 h in a parallel-‍plate shear flow system. (B) Macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear, or just incubated with media (Unstim.). Representative Western blots of PLXND1, iNOS (M1 macrophage marker) and GAPDH in macrophages are shown (C, D). PLXND1 and iNOS protein expressions were quantitatively analyzed from (B). These results show that oscillatory shear stress induced M1 macrophage polarization and increased PLXND1 expression at the same time. (E) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to laminar shear or oscillatory shear. The M1 macrophage marker, iNOS was detected by WB and quantification of WB in (F). Knockdown of PLXND1 attenuates M1 macrophage polarization induced by oscillatory shear stress. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. LS, laminar shear. OS, oscillatory shear.

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Shear, Derivative Assay, Cell Culture, Incubation, Western Blot, Marker, Expressing, Transfection, Control, Knockdown

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Oscillatory shear enhances M1 polarization by SEMA3E-PLXND1. (A) Immunofluorescence staining of bifurcation lesions was performed with iNOS (red), F4/80 (green), PLXND1 (pink), SEMA3E (green blue) and DAPI (blue). Representative images are shown on the left (scale bar, 100 μm) and enlarged images of inserts are shown on the right (scale bar, 20 μm). (B) THP-1-derived macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear in treatment with or without 500 ng/mL recombinant SEMA3E (rSEMA3E). Immunoblot analysis of iNOS are shown. (C) Quantitative analysis showed SEMA3E enhanced M1 polarization under oscillatory shear. (D) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to oscillatory shear in treatment with rSEMA3E. The expression of iNOS was analyzed by WB. (E) Quantitative analysis showed iNOS expression was significantly lower in sh-PLXND1 cells. Data are shown as the mean ± SEM. n = 4. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests or unpaired two-tailed Student's t tests. LS, laminar shear. OS, oscillatory shear. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Shear, Immunofluorescence, Staining, Derivative Assay, Cell Culture, Recombinant, Western Blot, Transfection, Control, Expressing, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: M1 macrophages express more PLXND1 than M2 macrophages in bifurcation lesions of ApoE −/− mice after 10 weeks and 20 weeks of high fat diet. (A) Adjacent sections of carotid bifurcation lesions were used to examine M1 and M2 macrophages by immunofluorescence staining separately. Immunofluorescence staining was performed with iNOS (green) or ARG1 (green), F4/80 (red), PLXND1 (pink), and DAPI (blue). Representative images are shown on the top (scale bar, 100 μm) and enlarged images of inserts are shown below (scale bar, 20 μm). (B) Intima PLXND1 + iNOS + F4/80 + M1 macrophages and PLXND1 + ARG1 + F4/80 + M2 macrophages were presented as number each slice. PLXND1 was expressed more in M1 macrophages rather than M2 macrophages. n = 4. ** P < 0.01, paired two-tailed t tests. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 expression level and location in process of atherosclerosis at bifurcations are evaluated by three-dimensional reconstruction. (A) 3D projections of PLXND1 (green) and iNOS (red) with autofluorescence at carotid bifurcations were depicted during atherosclerosis development by tissue clearing and light-sheet imaging. Representative images are shown (scale bar, 100 μm). Enlarged images of inserts are shown at right (scale bar, 10 μm). (B) Quantitative analyses of PLXND1 fluorescence of plaques show PLXND1 expression level increased by high-fat diet over time ( n = 4 mice; two carotid arteries/mice). (C) Quantitative analyses of iNOS fluorescence of plaques show iNOS expression level increased by high fat diet over time ( n = 4 mice; two carotid arteries/mice). (D) Co-localization of PLXND1 with iNOS in plaques at bifurcations was quantitatively analyzed ( n = 4 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. * P < 0.05, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Expressing, Imaging, Fluorescence

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Carotid bifurcation lesions are detected by probes targeting PLXND1. (A) Fluorescence imaging of isolated arteries from ApoE −/− mice were performed after intravenous injection of A12-Cy5, the molecular probes specific for PLXND1. A12-Cy5 showed stronger signals in carotid bifurcation lesions than common carotid arteries. (B) Arteries were conducted tissue clearing and light-sheet imaging. The images show that fluorescent probes accumulate inside the plaques (scale bar, 1 mm). (C) The ratio of fluorescent intensity between carotid bifurcation and common carotid artery were quantitatively analyzed at HFD 0w, 10w, 20w ( n = 3 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet.

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Fluorescence, Imaging, Isolation, Injection

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates LPS/IFN-γ-induced M1 macrophages polarization. (A) THP-1 cells were differentiated into macrophages, and then polarized to M1 or M2 macrophages by LPS/IFN-γ or IL-‍4/IL-13. Representative Western blots of PLXND1, iNOS (M1 marker), CD206 (M2 marker), CD163 (M2 marker), ARG1 (M2 marker) and GAPDH in M0, M1 and M2 type macrophages are shown. (B) Representative Western blots of PLXND1, p-STAT1/STAT1, p-STAT6/STAT6 and GAPDH in M0, M1 and M2 type macrophages are shown. (C) Quantitative analysis of PLXND1 protein expression demonstrates the highest level of PLXND1 protein in M1 macrophage. (D) THP-1 cells were transfected with shRNA to knockdown PLXND1. The knockdown efficiency was verified by western blotting (WB) and quantification of WB in (E). (F) THP-1 cells were transfected with sh-Control or sh-PLXND1 and further differentiated into macrophages via PMA. THP-1-derived macrophages were then incubated with or without LPS/IFN-γ. Representative Western blots of PLXND1, iNOS, and GAPDH are shown. (G) iNOS protein expression was quantitatively analyzed. Knockdown of PLXND1 attenuates the induction of iNOS, the M1 polarization marker in macrophages treated with LPS/IFN-γ. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Bonferroni's post-hoc test or unpaired two-tailed Student's t tests.

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Western Blot, Marker, Expressing, Transfection, shRNA, Derivative Assay, Incubation, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates M1 macrophages polarization induced by oscillatory shear stress. (A) A schematic drawing of the experiment is outlined. THP-1-derived macrophages were treated with oxLDL and co-cultured with HUVECs exposed to laminar shear or oscillatory shear for 24 h in a parallel-‍plate shear flow system. (B) Macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear, or just incubated with media (Unstim.). Representative Western blots of PLXND1, iNOS (M1 macrophage marker) and GAPDH in macrophages are shown (C, D). PLXND1 and iNOS protein expressions were quantitatively analyzed from (B). These results show that oscillatory shear stress induced M1 macrophage polarization and increased PLXND1 expression at the same time. (E) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to laminar shear or oscillatory shear. The M1 macrophage marker, iNOS was detected by WB and quantification of WB in (F). Knockdown of PLXND1 attenuates M1 macrophage polarization induced by oscillatory shear stress. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. LS, laminar shear. OS, oscillatory shear.

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Shear, Derivative Assay, Cell Culture, Incubation, Western Blot, Marker, Expressing, Transfection

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Oscillatory shear enhances M1 polarization by SEMA3E-PLXND1. (A) Immunofluorescence staining of bifurcation lesions was performed with iNOS (red), F4/80 (green), PLXND1 (pink), SEMA3E (green blue) and DAPI (blue). Representative images are shown on the left (scale bar, 100 μm) and enlarged images of inserts are shown on the right (scale bar, 20 μm). (B) THP-1-derived macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear in treatment with or without 500 ng/mL recombinant SEMA3E (rSEMA3E). Immunoblot analysis of iNOS are shown. (C) Quantitative analysis showed SEMA3E enhanced M1 polarization under oscillatory shear. (D) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to oscillatory shear in treatment with rSEMA3E. The expression of iNOS was analyzed by WB. (E) Quantitative analysis showed iNOS expression was significantly lower in sh-PLXND1 cells. Data are shown as the mean ± SEM. n = 4. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests or unpaired two-tailed Student's t tests. LS, laminar shear. OS, oscillatory shear. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies were anti-PLXND1 (NBP1-33634, Novus), anti-SEMA3E (AF3239, R&D Systems), anti-iNOS (ab283655, Abcam), anti-CD206 (ab125028, Abcam), anti-CD163 (ab156769, Abcam), anti-Liver Arginase (ab133543, Abcam), anti-STAT1 (phospho Y701) (ab109457, Abcam), anti-STAT1 (ab109320, Abcam), anti-STAT6 (phospho Y641) (ab263947, Abcam), anti-STAT6 (ab32520, Abcam) and anti-GAPDH (ab9485, Abcam) acted as an internal control.

Techniques: Shear, Immunofluorescence, Staining, Derivative Assay, Cell Culture, Recombinant, Western Blot, Transfection, Expressing, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: M1 macrophages express more PLXND1 than M2 macrophages in bifurcation lesions of ApoE −/− mice after 10 weeks and 20 weeks of high fat diet. (A) Adjacent sections of carotid bifurcation lesions were used to examine M1 and M2 macrophages by immunofluorescence staining separately. Immunofluorescence staining was performed with iNOS (green) or ARG1 (green), F4/80 (red), PLXND1 (pink), and DAPI (blue). Representative images are shown on the top (scale bar, 100 μm) and enlarged images of inserts are shown below (scale bar, 20 μm). (B) Intima PLXND1 + iNOS + F4/80 + M1 macrophages and PLXND1 + ARG1 + F4/80 + M2 macrophages were presented as number each slice. PLXND1 was expressed more in M1 macrophages rather than M2 macrophages. n = 4. ** P < 0.01, paired two-tailed t tests. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 expression level and location in process of atherosclerosis at bifurcations are evaluated by three-dimensional reconstruction. (A) 3D projections of PLXND1 (green) and iNOS (red) with autofluorescence at carotid bifurcations were depicted during atherosclerosis development by tissue clearing and light-sheet imaging. Representative images are shown (scale bar, 100 μm). Enlarged images of inserts are shown at right (scale bar, 10 μm). (B) Quantitative analyses of PLXND1 fluorescence of plaques show PLXND1 expression level increased by high-fat diet over time ( n = 4 mice; two carotid arteries/mice). (C) Quantitative analyses of iNOS fluorescence of plaques show iNOS expression level increased by high fat diet over time ( n = 4 mice; two carotid arteries/mice). (D) Co-localization of PLXND1 with iNOS in plaques at bifurcations was quantitatively analyzed ( n = 4 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. * P < 0.05, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems).

Techniques: Expressing, Imaging, Fluorescence

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Carotid bifurcation lesions are detected by probes targeting PLXND1. (A) Fluorescence imaging of isolated arteries from ApoE −/− mice were performed after intravenous injection of A12-Cy5, the molecular probes specific for PLXND1. A12-Cy5 showed stronger signals in carotid bifurcation lesions than common carotid arteries. (B) Arteries were conducted tissue clearing and light-sheet imaging. The images show that fluorescent probes accumulate inside the plaques (scale bar, 1 mm). (C) The ratio of fluorescent intensity between carotid bifurcation and common carotid artery were quantitatively analyzed at HFD 0w, 10w, 20w ( n = 3 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet.

Article Snippet: For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems).

Techniques: Fluorescence, Imaging, Isolation, Injection

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates LPS/IFN-γ-induced M1 macrophages polarization. (A) THP-1 cells were differentiated into macrophages, and then polarized to M1 or M2 macrophages by LPS/IFN-γ or IL-‍4/IL-13. Representative Western blots of PLXND1, iNOS (M1 marker), CD206 (M2 marker), CD163 (M2 marker), ARG1 (M2 marker) and GAPDH in M0, M1 and M2 type macrophages are shown. (B) Representative Western blots of PLXND1, p-STAT1/STAT1, p-STAT6/STAT6 and GAPDH in M0, M1 and M2 type macrophages are shown. (C) Quantitative analysis of PLXND1 protein expression demonstrates the highest level of PLXND1 protein in M1 macrophage. (D) THP-1 cells were transfected with shRNA to knockdown PLXND1. The knockdown efficiency was verified by western blotting (WB) and quantification of WB in (E). (F) THP-1 cells were transfected with sh-Control or sh-PLXND1 and further differentiated into macrophages via PMA. THP-1-derived macrophages were then incubated with or without LPS/IFN-γ. Representative Western blots of PLXND1, iNOS, and GAPDH are shown. (G) iNOS protein expression was quantitatively analyzed. Knockdown of PLXND1 attenuates the induction of iNOS, the M1 polarization marker in macrophages treated with LPS/IFN-γ. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Bonferroni's post-hoc test or unpaired two-tailed Student's t tests.

Article Snippet: For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems).

Techniques: Western Blot, Marker, Expressing, Transfection, shRNA, Knockdown, Control, Derivative Assay, Incubation, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates M1 macrophages polarization induced by oscillatory shear stress. (A) A schematic drawing of the experiment is outlined. THP-1-derived macrophages were treated with oxLDL and co-cultured with HUVECs exposed to laminar shear or oscillatory shear for 24 h in a parallel-‍plate shear flow system. (B) Macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear, or just incubated with media (Unstim.). Representative Western blots of PLXND1, iNOS (M1 macrophage marker) and GAPDH in macrophages are shown (C, D). PLXND1 and iNOS protein expressions were quantitatively analyzed from (B). These results show that oscillatory shear stress induced M1 macrophage polarization and increased PLXND1 expression at the same time. (E) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to laminar shear or oscillatory shear. The M1 macrophage marker, iNOS was detected by WB and quantification of WB in (F). Knockdown of PLXND1 attenuates M1 macrophage polarization induced by oscillatory shear stress. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. LS, laminar shear. OS, oscillatory shear.

Article Snippet: For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems).

Techniques: Shear, Derivative Assay, Cell Culture, Incubation, Western Blot, Marker, Expressing, Transfection, Control, Knockdown

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Oscillatory shear enhances M1 polarization by SEMA3E-PLXND1. (A) Immunofluorescence staining of bifurcation lesions was performed with iNOS (red), F4/80 (green), PLXND1 (pink), SEMA3E (green blue) and DAPI (blue). Representative images are shown on the left (scale bar, 100 μm) and enlarged images of inserts are shown on the right (scale bar, 20 μm). (B) THP-1-derived macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear in treatment with or without 500 ng/mL recombinant SEMA3E (rSEMA3E). Immunoblot analysis of iNOS are shown. (C) Quantitative analysis showed SEMA3E enhanced M1 polarization under oscillatory shear. (D) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to oscillatory shear in treatment with rSEMA3E. The expression of iNOS was analyzed by WB. (E) Quantitative analysis showed iNOS expression was significantly lower in sh-PLXND1 cells. Data are shown as the mean ± SEM. n = 4. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests or unpaired two-tailed Student's t tests. LS, laminar shear. OS, oscillatory shear. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For immunofluorescence staining, the following primary antibodies were used: PLXND1 (NBP1-33634, Novus), iNOS (ab283655, Abcam), Liver Arginase (ab239731, Abcam), F4/80 (ab300421, Abcam) and SEMA3E (AF3239, R&D Systems).

Techniques: Shear, Immunofluorescence, Staining, Derivative Assay, Cell Culture, Recombinant, Western Blot, Transfection, Control, Expressing, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: M1 macrophages express more PLXND1 than M2 macrophages in bifurcation lesions of ApoE −/− mice after 10 weeks and 20 weeks of high fat diet. (A) Adjacent sections of carotid bifurcation lesions were used to examine M1 and M2 macrophages by immunofluorescence staining separately. Immunofluorescence staining was performed with iNOS (green) or ARG1 (green), F4/80 (red), PLXND1 (pink), and DAPI (blue). Representative images are shown on the top (scale bar, 100 μm) and enlarged images of inserts are shown below (scale bar, 20 μm). (B) Intima PLXND1 + iNOS + F4/80 + M1 macrophages and PLXND1 + ARG1 + F4/80 + M2 macrophages were presented as number each slice. PLXND1 was expressed more in M1 macrophages rather than M2 macrophages. n = 4. ** P < 0.01, paired two-tailed t tests. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Samples were next incubated with primary antibodies for PLXND1 (NBP1-33634, Novus) and iNOS (14-5920-82, Invitrogen) in PTwH and 5% DMSO at 37 °C for 3 d. Following washing 10 times with PTwH at each hour, the tissues were stained with Alexa Fluor 647-conjugated and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) for 3 d and finally washed with PTwH for 3 h 5 times.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 expression level and location in process of atherosclerosis at bifurcations are evaluated by three-dimensional reconstruction. (A) 3D projections of PLXND1 (green) and iNOS (red) with autofluorescence at carotid bifurcations were depicted during atherosclerosis development by tissue clearing and light-sheet imaging. Representative images are shown (scale bar, 100 μm). Enlarged images of inserts are shown at right (scale bar, 10 μm). (B) Quantitative analyses of PLXND1 fluorescence of plaques show PLXND1 expression level increased by high-fat diet over time ( n = 4 mice; two carotid arteries/mice). (C) Quantitative analyses of iNOS fluorescence of plaques show iNOS expression level increased by high fat diet over time ( n = 4 mice; two carotid arteries/mice). (D) Co-localization of PLXND1 with iNOS in plaques at bifurcations was quantitatively analyzed ( n = 4 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. * P < 0.05, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Samples were next incubated with primary antibodies for PLXND1 (NBP1-33634, Novus) and iNOS (14-5920-82, Invitrogen) in PTwH and 5% DMSO at 37 °C for 3 d. Following washing 10 times with PTwH at each hour, the tissues were stained with Alexa Fluor 647-conjugated and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) for 3 d and finally washed with PTwH for 3 h 5 times.

Techniques: Expressing, Imaging, Fluorescence

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Carotid bifurcation lesions are detected by probes targeting PLXND1. (A) Fluorescence imaging of isolated arteries from ApoE −/− mice were performed after intravenous injection of A12-Cy5, the molecular probes specific for PLXND1. A12-Cy5 showed stronger signals in carotid bifurcation lesions than common carotid arteries. (B) Arteries were conducted tissue clearing and light-sheet imaging. The images show that fluorescent probes accumulate inside the plaques (scale bar, 1 mm). (C) The ratio of fluorescent intensity between carotid bifurcation and common carotid artery were quantitatively analyzed at HFD 0w, 10w, 20w ( n = 3 mice; two carotid arteries/mice). Data are shown as the mean ± SEM. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. AF, autofluorescence. HFD, high-fat diet.

Article Snippet: Samples were next incubated with primary antibodies for PLXND1 (NBP1-33634, Novus) and iNOS (14-5920-82, Invitrogen) in PTwH and 5% DMSO at 37 °C for 3 d. Following washing 10 times with PTwH at each hour, the tissues were stained with Alexa Fluor 647-conjugated and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) for 3 d and finally washed with PTwH for 3 h 5 times.

Techniques: Fluorescence, Imaging, Isolation, Injection

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates LPS/IFN-γ-induced M1 macrophages polarization. (A) THP-1 cells were differentiated into macrophages, and then polarized to M1 or M2 macrophages by LPS/IFN-γ or IL-‍4/IL-13. Representative Western blots of PLXND1, iNOS (M1 marker), CD206 (M2 marker), CD163 (M2 marker), ARG1 (M2 marker) and GAPDH in M0, M1 and M2 type macrophages are shown. (B) Representative Western blots of PLXND1, p-STAT1/STAT1, p-STAT6/STAT6 and GAPDH in M0, M1 and M2 type macrophages are shown. (C) Quantitative analysis of PLXND1 protein expression demonstrates the highest level of PLXND1 protein in M1 macrophage. (D) THP-1 cells were transfected with shRNA to knockdown PLXND1. The knockdown efficiency was verified by western blotting (WB) and quantification of WB in (E). (F) THP-1 cells were transfected with sh-Control or sh-PLXND1 and further differentiated into macrophages via PMA. THP-1-derived macrophages were then incubated with or without LPS/IFN-γ. Representative Western blots of PLXND1, iNOS, and GAPDH are shown. (G) iNOS protein expression was quantitatively analyzed. Knockdown of PLXND1 attenuates the induction of iNOS, the M1 polarization marker in macrophages treated with LPS/IFN-γ. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Bonferroni's post-hoc test or unpaired two-tailed Student's t tests.

Article Snippet: Samples were next incubated with primary antibodies for PLXND1 (NBP1-33634, Novus) and iNOS (14-5920-82, Invitrogen) in PTwH and 5% DMSO at 37 °C for 3 d. Following washing 10 times with PTwH at each hour, the tissues were stained with Alexa Fluor 647-conjugated and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) for 3 d and finally washed with PTwH for 3 h 5 times.

Techniques: Western Blot, Marker, Expressing, Transfection, shRNA, Knockdown, Control, Derivative Assay, Incubation, Two Tailed Test

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: PLXND1 regulates M1 macrophages polarization induced by oscillatory shear stress. (A) A schematic drawing of the experiment is outlined. THP-1-derived macrophages were treated with oxLDL and co-cultured with HUVECs exposed to laminar shear or oscillatory shear for 24 h in a parallel-‍plate shear flow system. (B) Macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear, or just incubated with media (Unstim.). Representative Western blots of PLXND1, iNOS (M1 macrophage marker) and GAPDH in macrophages are shown (C, D). PLXND1 and iNOS protein expressions were quantitatively analyzed from (B). These results show that oscillatory shear stress induced M1 macrophage polarization and increased PLXND1 expression at the same time. (E) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to laminar shear or oscillatory shear. The M1 macrophage marker, iNOS was detected by WB and quantification of WB in (F). Knockdown of PLXND1 attenuates M1 macrophage polarization induced by oscillatory shear stress. Data are shown as the mean ± SEM. n = 4. * P < 0.05, ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests. LS, laminar shear. OS, oscillatory shear.

Article Snippet: Samples were next incubated with primary antibodies for PLXND1 (NBP1-33634, Novus) and iNOS (14-5920-82, Invitrogen) in PTwH and 5% DMSO at 37 °C for 3 d. Following washing 10 times with PTwH at each hour, the tissues were stained with Alexa Fluor 647-conjugated and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) for 3 d and finally washed with PTwH for 3 h 5 times.

Techniques: Shear, Derivative Assay, Cell Culture, Incubation, Western Blot, Marker, Expressing, Transfection, Control, Knockdown

Journal: Heliyon

Article Title: Plexin D1 mediates disturbed flow-induced M1 macrophage polarization in atherosclerosis

doi: 10.1016/j.heliyon.2023.e17314

Figure Lengend Snippet: Oscillatory shear enhances M1 polarization by SEMA3E-PLXND1. (A) Immunofluorescence staining of bifurcation lesions was performed with iNOS (red), F4/80 (green), PLXND1 (pink), SEMA3E (green blue) and DAPI (blue). Representative images are shown on the left (scale bar, 100 μm) and enlarged images of inserts are shown on the right (scale bar, 20 μm). (B) THP-1-derived macrophages were co-cultured with HUVECs exposed to laminar shear or oscillatory shear in treatment with or without 500 ng/mL recombinant SEMA3E (rSEMA3E). Immunoblot analysis of iNOS are shown. (C) Quantitative analysis showed SEMA3E enhanced M1 polarization under oscillatory shear. (D) THP-1-derived macrophages transfected with sh-Control or sh-PLXND1 were co-cultured with HUVECs exposed to oscillatory shear in treatment with rSEMA3E. The expression of iNOS was analyzed by WB. (E) Quantitative analysis showed iNOS expression was significantly lower in sh-PLXND1 cells. Data are shown as the mean ± SEM. n = 4. ** P < 0.01, **** P < 0.0001, one-way ANOVA with Bonferroni's post-hoc tests or unpaired two-tailed Student's t tests. LS, laminar shear. OS, oscillatory shear. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Samples were next incubated with primary antibodies for PLXND1 (NBP1-33634, Novus) and iNOS (14-5920-82, Invitrogen) in PTwH and 5% DMSO at 37 °C for 3 d. Following washing 10 times with PTwH at each hour, the tissues were stained with Alexa Fluor 647-conjugated and Alexa Fluor 568-conjugated secondary antibodies (Invitrogen) for 3 d and finally washed with PTwH for 3 h 5 times.

Techniques: Shear, Immunofluorescence, Staining, Derivative Assay, Cell Culture, Recombinant, Western Blot, Transfection, Control, Expressing, Two Tailed Test