Therapeutic CCR2 Blockade Prevents Inflammation and Alleviates Myxomatous Valve Disease in Marfan Syndrome

Figure 1. CCR2 Deficiency Restores Immune Cells and ECM Organization in MFS Valves (A) CD45+ leukocytes (arrows, green) are significantly increased in mitral valves of 2-month-old Fbn1C1039G/+ mice by immunofluorescence. (B) Immunofluorescence with collagen hybridizing peptide (CHP) (arrowheads, green), collagen I (Col I) (asterisks, red), hyaluronic acid binding protein (HABP) (white) show extracellular matrix (ECM) abnormalities in 2-month-old Fbn1C1039G/+ mice with Marfan syndrome (MFS). (C) CD45+ leukocytes (green) and red fluorescent protein (RFP+) cells (red) are prevalent in mitral valves of Fbn1C1039G/+; CCR2RFP/+ mice vs controls. Fbn1C1039G/+; CCR2RFP/RFP mice have a reduction of both CD45+ leukocytes and RFP+ cells (arrowheads, double positive cells) relative to Fbn1C1039G/+; CCR2RFP/+ controls. (D) Pathological ECM remodeling indicated by immunofluorescence with CHP (green, arrowheads indicating collagen breaks), Col I (red), and HABP (white) was improved by C-C chemokine receptor type 2 (CCR2) deficiency in Fbn1C1039G/+ mice. Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI). Scale bar = 100 μm. MVAL = mitral valve anterior leaflet; WT = wild-type.

Figure 2. CCR2 Knockout Inhibits Inflammation in Fbn1C1039G/+ Mitral Valves RNA sequencing (RNAseq) was performed from the mitral valves of 2-month-old Fbn1C1039G/+ mice with CCR2RFP/RFP (FhetCko) compared to CCR2RFP/+ (Chet) and Fbn1C1039G/+; CCR2RFP/+ (FhetChet) mice. (A) Venn diagram shows the common 56 differentially expressed genes between FhetChet vs Chet upregulated and FhetCko vs FhetChet downregulated genes. (B) The heatmap of the common differentially expressed genes in Chet, FhetChet, and FhetCko groups. (C) Gene ontology (GO) term analysis of the common genes by DAVID (Database for Annotation, Visualization, and Integrated Discovery). N = 4. BP = biological process; Chet = CCR2RFP/+, FhetChet = Fbn1C1039G/+; CCR2RFP/+, FhetCko = Fbn1C1039G/+; CCR2RFP/RFP; MF = molecular function; other abbreviation as in Figure 1.

Figure 3. Loss of CCR2 Leads to Reduced Inflammatory Protease Expression (A) Immunofluorescence indicated increased protein expression of Mcpt4 (green) and Cpa3 (red) specifically from mast cells at the thickening area of MFS valves which was restored by CCR2 knockout. Scale bar = 100 μm. (B) Quantification of immunofluorescence showed Mcpt4 and Cpa3 pixel area was significantly decreased by CCR2 deficiency. (C) Immunofluorescence indicated that Aebp1 (red) was not only expressed in CD45+ immune cells, but was also expressed in other cell types. The increased expression of Aebp1 (red) in MFS valves was inhibited by CCR2 deficiency. The nuclei were counterstained with DAPI. Scale bar = 100 μm. (D) Quantification of immunofluorescence showed Aebp1 pixel area and cell number of Aebp1+ CD45+ immune cells were reduced by CCR2 deficiency. One-way analysis of variance (ANOVA) with Tukey’s post hoc test is used and data are represented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Aebp1 = adipocyte enhancer binding protein 1; Cko = CCR2RFP/RFP; Cpa3 = carboxypeptidase A3; Mcpt4 = mast cell protease 4; other abbreviations as in Figures 1 and 2.

Figure 4. RS504393 Treatment Decreases CD45+ Immune Cells and MHCII+ Macrophages in Fbn1C1039G/+ Valves (A) Chemical structure of CCR2 specific inhibitor RS504393. (B) Schematic of RS504393 intraperitoneal daily injection treatment strategy in 1-month-old WT and Fbn1C1039G/+ mice for 1 month. (C) Representative images of immunofluorescence with CD45 (arrows, green) in mitral valves of WT and Fbn1C1039G/+ mice treated with dimethyl sulfoxide (DMSO) (vehicle) or RS504393. Scale bar = 100 μm. (D) Quantification of number of CD45+ cells. (E) Representative images of immunofluorescence detection of CD206+ (green) and MHCII+ (arrows, red) macrophage subpopulations in mitral valves of WT and Fbn1C1039G/+ mice treated with vehicle or RS504393. Scale bar = 100 μm. (F) Quantification of cell number of macrophage subpopulations. Two-way ANOVA with Tukey’s post hoc test is used and data are represented as mean ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001. MVD = myxomatous valve degeneration; MHCII = major histocompatibility complex class II; other abbreviations as in Figures 1, 2, and 3.

Figure 5. CCR2 Inhibition Prevents Pathological ECM Remodeling in Fbn1C1039G/+ Valves (A) Immunofluorescence with CHP (green, arrowheads indicating collagen breaks) showed collagen remodeling in mitral valves of Fbn1C1039G/+ mice treated with DMSO (vehicle) or RS504393. Scale bar = 100 μm. (B) Quantification of collagen fiber breakdown. (C) Col I (green) immunostaining showed collagen I deposition. (D) Quantification of collagen I pixel area in mitral valves. (E) Proteoglycan accumulation is shown in vehicle-treated Fbn1C1039G/+ mice compared with control by immunofluorescence with HABP (red). RS504393 prevented increased proteoglycan accumulation in myxomatous valves. Scale bar = 100 μm. (F) Quantification of HABP pixel area. (G) Representative Movat pentachrome staining of mitral valves of Fbn1C1039G/+ mice treated with vehicle or RS504393 (collagen, yellow; proteoglycan, blue; elastin, black; muscle, red). Scale bar = 100 μm. (H) Quantification of mitral valve thickness. Two-way ANOVA with Tukey’s post hoc test is used and data are represented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 1, 2, 3, and 4.

Figure 6. RS504393 Treatment Prevents Pathologic Induction of Inflammatory Proteases in Fbn1C1039G/+ Valves (A) Immunofluorescence indicated protein expression of the chymase Mcpt4 (green) and carboxypeptidase Cpa3 (red) in mitral valves of Fbn1C1039G/+ mice treated with CCR2 inhibitor RS504393 compared to DMSO vehicle controls. Mcpt4 and Cpa3 specifically released from mast cells were increased at the thickened area of mitral valves of vehicle-treated Fbn1C1039G/+ mice where pathologic collagen dysregulation and proteoglycan accumulation occur. RS504393 treatment restores their levels and distribution. (B) Quantification of pixel area of Mcpt4 and Cpa3. (C) Immunofluorescence indicated increased protein levels of Aebp1 (red) in regions of pathologic ECM remodeling of Fbn1C1039G/+ valves was inhibited by RS504393 treatment. The nuclei were counterstained with DAPI. Scale bar = 100 μm. (D) Quantification of Aebp1 pixel area and number of Aebp1+ CD45+ cells. Two-way ANOVA with Tukey’s post hoc test is used and data are represented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01. Abbreviations as in Figures 1, 2, 3, and 4.

Figure 7. CCR2 Inhibition Prevents Myxomatous Valve Degeneration In 6-Month-Old MFS Mice (A) Schematic of RS504393 intraperitoneal daily injection into 6-month-old WT and Fbn1C1039G/+ mice for 1 month. (B) Representative Movat pentachrome staining of mitral valves of WT and Fbn1C1039G/+ mice treated with vehicle or RS504393 (collagen, yellow; proteoglycan, blue; elastin, black; muscle, red). Scale bar = 100 μm. (C) Quantification of mitral valve thickness. Two-way ANOVA with Tukey’s post hoc test is used and data are represented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01. Abbreviations as in Figures 1, 2, 3, and 4.