Figure 6.
17R-RvD1 protects against the H/R activation of profibrotic pathways in sickle cell mice. (A) Immunoblot analysis using specific antibodies against p-FGF-R, FGF-R, p-PDGF-R, and PDGF-R in the hearts of AA and SS mice under normoxia, treated with vehicle or 17R-RvD1 (100 ng), and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). One representative gel from 4 gels with similar results is shown; 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. Densitometric analysis immunoblots are shown in supplemental Figure 12A. (B) IP of the hearts of AA and SS mice, treated similar to panel A, using specific IP: PY, revealed with specific anti–TGF-β Rec antibody (75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel). GAPDH in WCL is used as loading controls. One representative gel from 4 others with similar results is presented. Densitometric analysis of immunoblots is shown (lower panel). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (C) Immunoblot analysis using specific antibodies against phosphorylated Smad2 (p-Smad2), Smad2, p-Smad3, Smad3, Smad4, and Smad7 in the hearts of AA and SS mice under normoxia, treated with vehicle or 17R-RvD1 (100 ng), and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). One representative gel from 4 gels with similar results is shown. A total of 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. Densitometric analysis immunoblots are shown in supplemental Figure 12C. (D) Immunoblot analysis using specific antibodies against HIF1α and HIF2 in the hearts of AA and SS mice treated similar to panel C. One representative gel from 4 gels with similar results is shown; 50 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (E) Immunoblot analysis (left) using specific antibodies against phosphorylated VEGF receptor (p-VEGF-R), VEGF-R, angiopoietin-1 (Ang 1), and Ang 2 in the hearts of AA and SS mice treated similar to panel C. One representative gel from 4 gels with similar results is shown; 75 μg/μL of protein loaded on a 10% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. Densitometric analysis of immunoblots is shown in supplemental Figure 13A. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia). °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). Representative merged immunofluorescence staining of VEG-FR on small and large CD31+ vascular endothelial cells in the hearts of SS mice undergoing H/R and treated with vehicle or 17R-RvD1 (right). Arrow denotes a large blood vessel staining positive for VEGF-R. Nuclei were stained with DAPI. Separate staining is shown in supplemental Figure 13B. Wb, Western blot.

17R-RvD1 protects against the H/R activation of profibrotic pathways in sickle cell mice. (A) Immunoblot analysis using specific antibodies against p-FGF-R, FGF-R, p-PDGF-R, and PDGF-R in the hearts of AA and SS mice under normoxia, treated with vehicle or 17R-RvD1 (100 ng), and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). One representative gel from 4 gels with similar results is shown; 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. Densitometric analysis immunoblots are shown in supplemental Figure 12A. (B) IP of the hearts of AA and SS mice, treated similar to panel A, using specific IP: PY, revealed with specific anti–TGF-β Rec antibody (75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel). GAPDH in WCL is used as loading controls. One representative gel from 4 others with similar results is presented. Densitometric analysis of immunoblots is shown (lower panel). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (C) Immunoblot analysis using specific antibodies against phosphorylated Smad2 (p-Smad2), Smad2, p-Smad3, Smad3, Smad4, and Smad7 in the hearts of AA and SS mice under normoxia, treated with vehicle or 17R-RvD1 (100 ng), and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). One representative gel from 4 gels with similar results is shown. A total of 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. Densitometric analysis immunoblots are shown in supplemental Figure 12C. (D) Immunoblot analysis using specific antibodies against HIF1α and HIF2 in the hearts of AA and SS mice treated similar to panel C. One representative gel from 4 gels with similar results is shown; 50 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. Densitometric analysis of immunoblots is shown in the lower panel. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (E) Immunoblot analysis (left) using specific antibodies against phosphorylated VEGF receptor (p-VEGF-R), VEGF-R, angiopoietin-1 (Ang 1), and Ang 2 in the hearts of AA and SS mice treated similar to panel C. One representative gel from 4 gels with similar results is shown; 75 μg/μL of protein loaded on a 10% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. Densitometric analysis of immunoblots is shown in supplemental Figure 13A. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia). °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). Representative merged immunofluorescence staining of VEG-FR on small and large CD31+ vascular endothelial cells in the hearts of SS mice undergoing H/R and treated with vehicle or 17R-RvD1 (right). Arrow denotes a large blood vessel staining positive for VEGF-R. Nuclei were stained with DAPI. Separate staining is shown in supplemental Figure 13B. Wb, Western blot.

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