17(R)-Resolvin D1 protects against sickle cell-related inflammatory cardiomyopathy in humanized mice

Abstract

Cardiovascular disease has been recognized as the main cause of death in adults with sickle cell disease (SCD). Although the exact mechanism linking SCD to cardiomyopathy remains elusive, a possible role of subclinical acute transient myocardial ischemia during acute sickle cell-related vaso-occlusive crises (VOCs) has been suggested. We approached SCD cardiomyopathy by integrated omics using humanized SS mice exposed to hypoxia/reoxygenation (H/R; 10 hours hypoxia followed by 3 hours reoxygenation) stress, mimicking acute VOCs. In sickle cell (SS) mice exposed to H/R, a neutrophil-driven cardiac hypertrophic response is initiated by cardiac proinflammatory pathways, intersecting proteins and micro RNA involved in profibrotic signaling. This response may be facilitated by local unresolved inflammation. We then examined the effect of 17(R)-resolvin D1 (17R-RvD1), a member of the specialized proresolving lipid mediator superfamily, administration on H/R-activated profibrotic and proangiogenic pathways. In SS mice, we found that 17R-RvD1 (1) modulates miRNAome; (2) prevents the activation of NF-κB p65; (3) protects against the H/R-induced activation of both platelet derived growth factor receptor and transforming growth factor (TGF)-β1/Smad2-3 canonical pathways; (4) reduces the expression of hypoxia-inducible factor-dependent proangiogenic signaling; and (5) decreases the H/R-induced proapoptotic cell signature. The protective role of 17R-RvD1 against H/R-induced maladaptive heart remodeling was supported by the reduction of galectin-3, procollagen C-proteinase enhancer-1, and endothelin-1 expression and perivascular fibrosis in SS mice at 3 days after H/R stress compared with vehicle-treated SS animals. Collectively, our data support the novel role of unresolved inflammation in pathologic heart remodeling in SCD mice in response to H/R stress. Our study provides new evidence for protective effects of 17R-RvD1 against SCD-related cardiovascular disease.

Graphical abstract

Figure 1.

In sickle cell mice, heart proteomic analysis reveals that H/R stress activates proinflammatory and profibrotic pathways. (A) Schematic diagram of the experimental plan used in this study. (B) Ingenuity pathway analysis (IPA) networks generated interrogating proteins identified as differentially expressed in SS hearts under H/R stress compared with AA under H/R. The following pathways were identified as affected by H/R in the hearts of SS mice vs healthy animals: (1) fibrosis of the heart; (2) hypertrophy of the heart; and (3) apoptosis of cardiomyocytes. (C) Immunoblot analysis using specific antibodies against phosphorylated Nrf2 (p-Nrf2) and Nrf2 in the hearts of AA and SS mice in normoxia (N) and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± standard error of the mean (SEM; n = 4). §P < .05 (compared with AA normoxia); ∗P < .05 (compared with normoxia by 1-way analysis of variance [ANOVA]). (D) Immunoblot analysis, using specific antibodies against phosphorylated FGF receptor (p-FGF-R), FGF-R, p-PDGF-R-B, and PDGFR-B, in the hearts of AA and SS mice. A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as mean ± SEM (n = 4). §P < .05 (compared with AA normoxia); ∗P < .05 (compared with normoxia by 1-way ANOVA). (E) Immunoprecipitation (IP) from the hearts of AA and SS mice, treated similar to panel D, using specific anti–phospho-tyrosine (PY) antibodies (IP: PY), revealed with specific anti–TGF-β receptor (TGF-β-Rec) antibody (75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel). GAPDH in whole cell lysate (WCL) is used as loading controls. One representative gel from 4 others with similar results is presented. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia by t test). TGF, transforming growth factor; Wb, Western blot.

Figure 2.

Hypoxia/reoxygenation stress modulates microRNAs in the hearts of sickle cell mice. (A) Relative expression of microRNAs from the hearts obtained from AA or SS mice under normoxia or H/R conditions. Expression of miRNAs was determined with quantitative polymerase chain reaction and normalized using U6SNRNA, RNU5G, RNU1A1, and SNORD61 as housekeeping small noncoding RNAs. Results are mean ± SEM from 3 separate mice. ∗P < .05; ∗∗P < .01 (1-way ANOVA). (B-C) Heat map and volcano plot showing fold change (FC) in the relative expression of miRNAs in the hearts of AA and SS mice under H/R compared with normoxia. Expression of microRNAs was determined (from n = 5 mice per condition). Dotted lines in the volcano plot represents cutoff values for significant (P < .05) differentially expressed (–0.58 > log₂ FC > 0.58) in SS hearts under H/R compared with AA hearts under H/R. (D) IPA networks generated interrogating proteins targets of differentially expressed miRNAs in SS hearts under H/R stress.

Figure 3.

In sickle cell mice, 17R-RvD1 preserves cardiac contractility and protects against H/R-induced neutrophil heart infiltration. (A) Fractional shortening (FS [%]) in SS mice under normoxia and exposed to H/R stress and treated with either vehicle or 17R-RvD1 (n = 3-4); P < .05 (compared with vehicle-treated H/R SS mice). (B) Serum Gal-3 from AA and SS mice under normoxia and treated with vehicle or 17R-RvD1. Data are presented as mean ± SEM (n = 5-9). ∗∗∗∗P < .005 (by unpaired t test with Welch correction). (C) Immunofluorescence expression of Gal-3 (red) in heart microsections from AA mice and SS mice exposed to H/R stress treated with either vehicle or 17R-RvD1. Nuclei (blue) were stained with DAPI. Quantification of Gal-3 was performed on 3 to 4 samples per group (6 × 400 field per sample) with ZEN 2.3 Software. Data are presented as mean ± SEM (n = 3-4). ∗P < .05; ∗∗∗P < .001 (by 1-way ANOVA). (D) Heart neutrophils infiltration identified, by flow cytometric analysis, as CD45⁺Ly6G⁺ cells from AA and SS mice under normoxia and 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). Data are presented as mean ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated H/R SS mice by 1-way ANOVA). (E) One representative image by immunomicroscopy (left) and quantification (right) of proresolving CD206-polarized intracardiac neutrophils (per mm² heart tissue) identified by double immunofluorescence for Ly6G and CD206 in SS mice exposed to H/R and treated with vehicle or 17R-RvD1. Data are presented as mean ± SEM (n = 3-4). °P < .05 (compared with vehicle-treated H/R SS mice by t test; size scale bar, 40 μm). MFI, mean fluorescence intensity.

Figure 4.

17R-RvD1 prevents the activation of NF-κB–dependent pathways and reduces NLRP3 inflammasome. (A) Activated phosphorylated NF-κB p65 (p-NF-κB p65) in heart cells identified with immunofluorescence staining (size scale bar, 20 μm) in SS mice exposed to H/R stress and treated with either vehicle or 17R-RvD1 (left). Data are presented as mean ± SEM (n = 5). °P < .05 (compared with vehicle-treated H/R SS mice by t test). Quantification of total NF-κB in heart cells is shown in supplemental Figure 8B. Immunoblot analysis (right), using specific antibodies against p-NF-κB p65 and NF-κB p65, of heart from AA and SS mice under normoxia and 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). A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (B) Immunoblot analysis, using specific antibodies against P-selectin, ET-1, and thromboxane synthase (TBXS), in the hearts of AA and SS mice treated. A total of 75 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (C) Immunoblot analysis, using specific antibodies against NLRP3, in the hearts of AA and SS mice, treated similar to panel B. A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (lower). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); °P < .05 (compared with vehicle-treated mice). Wb, Western blot.

Figure 5.

17R-RvD1 prevents H/R-induced activation of Nfr2 system and modulates miRNA related to proinflammatory and profibrotic pathways in the hearts of sickle cell mice. (A) Immunoblot analysis using specific antibodies against p-Nrf2 and Nrf2 in the hearts of AA and SS mice under normoxia and 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); 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. One representative gel from 4 gels with similar results is shown. Densitometric analysis of immunoblots is shown on the right. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice). (B) Immunoblot analysis using specific antibodies against HO-1 and Nqo1 in the hearts of AA and SS mice treated, similar to panel A; 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. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). 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 Prx2 and Prx3, in the hearts of AA and SS mice treated similar to panel A; 30 μg/μL of protein loaded on an 11% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia); §P < .05 (compared with AA normoxia); °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). (D) miRNAs regulated by 17R-RvD1 in the hearts of SS mice undergoing H/R. MicroRNA expression was determined using RNU5G, RNU1A1, and SNORD61 as housekeeping small noncoding RNAs. ∗P < .05; ∗∗P < .01 (1-way ANOVA). Wb, Western blot.

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.

Figure 7.

In sickle cell mice, 17R-RvD1 reduces proangiogenic signaling, prevents H/R-induced ER stress, and protects against proapoptotic H/R-induced signature. (A) Serum Gal-3 (left) and procollagen C-proteinase enhancer-1 (PCPE1) in SS mice treated with either vehicle or 17R-RvD1 at 3 days after H/R stress. Data are presented as mean ± SEM (n = 3-4). ∗P < .05; ∗∗∗P < .005 (by unpaired t test, with Welch correction). a-SMA (B) and Picrosirius Red (C) staining in cardiac slices from SS mice treated with either vehicle or 17R-RvD1 at 3 days after H/R stress. (D) Immunoblot analysis using specific antibodies against ATF6, GADD34, ATF4, and CHOP 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 of immunoblots is shown (right). 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 using specific antibodies against caspase-3 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. GAPDH serves as protein loading control. Densitometric analysis of immunoblots is shown (lower). Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia). °P < .05 (compared with vehicle-treated mice by 1-way ANOVA). Wb, Western blot.