The immunosuppressant FTY720 prolongs survival in a mouse model of diet-induced coronary atherosclerosis and myocardial infarction

Abstract

FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61(h/h)/SRB1(-/- mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg(-1)·d(-1)) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61(h/h)/SRB1(-/-) mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg(-1)·d(-1)). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-β and reduced IFN-γ expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-β and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.

Figure 1

Design of the mortality study (A). Design of the pathology study (B). High fat diet = High fat, high cholesterol diet.

Figure 2

FTY720-treatment in wildtype C57BL/6J mice improved recovery of left ventricular developed pressure (LVDP) (A) and reduced infarct size (B) after ex vivo ischemia/reperfusion (I/R) injury. The % at the end of the ischemia/reperfusion period was calculated relative to the baseline LVDP expressed in mmHg for each heart. Data are mean ± SEM, n=6/group, * P<0.05 vs control. FTY=FTY720; Con=Control. (C) Kaplan–Meier survival curve for ApoeR61^h/h/SRB1^–/– mice fed the HFD and treated or not with FTY720 (FTY720 and Control) for 4 weeks. The FTY720-treated mice exhibited a markedly lower mortality rate (P<0.02). HFD = high fat high cholesterol diet.

Figure 3

Echocardiographic measures of left ventricular performance. Ejection fraction (A) and fractional shortening (B), left ventricular end-diastolic volume (LVEDV) (C) and left ventricular end-systolic volume (LVESV) (D) in ApoeR61^h/h/SRB1^–/– mice fed the HFD and treated or not with FTY720 for 4 weeks. Data are mean ± SEM. * P<0.05 vs control. HFD = high fat high cholesterol diet.

Figure 4

(A and B) Plasma cholesterol levels (n=16-20 ApoeR61^h/h/SRB1^–/– mice/group) and lipoprotein profiles (n=3 pools with 3-7 mice/pool). Data are at 1wk pre-HFD and at 3.5wk after starting the HFD. Mice were fed FTY720 or not. As can be seen, the lipoprotein profiles are identical. Lipoprotein classes are indicated as VLDL (very low density lipoprotein), IDL/LDL (intermediate density lipoprotein / low density lipoprotein) and HDL (high density lipoprotein) (C) Plasma triglyceride levels obtained in the same mice also show no difference. Data are mean ± SEM. * P<0.001 (1wk pre-HFD vs 3.5wk HFD for both control and FTY720). 1wk pre-HFD = 1week ± FTY720-treatment on chow diet; 3.5wk HFD = 3.5 weeks ± FTY720-treatment with HFD feeding. HFD = high fat high cholesterol diet.

Figure 5

Representative images of aortic root lesions (A and B) and partially (C and D) or severely (E and F) occluded coronary arteries stained with Sirius red from ApoeR61^h/h/SRB1^–/– mice fed the HFD for 3.5 weeks with FTY720-treatment or not. (n=7-8 mice/group). Quantification of aortic root lesion area (G) and occluded coronary arteries (H). Black arrows identify the plaque accumulated in the vasculature. Data are mean ± SEM. NO = non occluded (0-5%); PO = partially occluded (5-50%); SO = severely occluded (50-95%); CO = completely occluded (95-100%).

Figure 6

Images of heart sections showing the left ventricular chamber stained with Sirius Red (A and B) and Oil red O (C and D) of ApoeR61^h/h/SRB1^–/– mice fed the HFD for 3.5 weeks with FTY720-treatment or not (n=7-8 mice/group). Quantification of collagen (E) and neutral lipid (F) deposited in the myocardium. Black arrows identify the area with clusters of collagen or neutral lipid. Data are mean ± SEM. ORO = oil red O

Figure 7

(A) Number of CD45⁺ leukocytes per μl of blood of ApoeR61^h/h/SRB1^–/– mice at 1wk pre-HFD and 3.5wk HFD time-points with FTY720-treatment or not. (B) Numbers of B cells, T cells, monocytes (mono) and neutrophils (neutro) per μl of blood at 1wk pre-HFD and 3.5wk HFD time-points. (C) Representative FACS plots of CD45+ blood leukocytes from ApoeR61^h/h/SRB1^–/– mice after 3.5 weeks of HFD feeding. Shown are gated populations: T cells (CD3⁺B220^–), B cells (CD3^–B220⁺), neutrophils (Ly6G⁺CD11b⁺) and monocytes (Ly6G^–CD11b⁺). (D) Numbers of CD4⁺ and CD8⁺ T cells per μl of blood after 3.5 weeks of HFD feeding. (E) Percentage of Foxp3⁺ T cells among CD4⁺ T cells in the circulation, LN and spleen after 3.5 weeks of HFD feeding. Data are mean ± SEM. * P<0.05, ** P<0.01 vs Control. 1wk pre-HFD = 1week chow diet with FTY720-treatment or not; 3.5wk HFD = 3.5 weeks HFD with FTY720-treatment or not; LN = lymph nodes. HFD = high fat high cholesterol diet.

Figure 8

(A) Relative mRNA expression levels of TGF-β and IFN-γ. (B) CD45⁺ leukocytes and CD4⁺ T cells in hearts from mice not fed the HFD and after 3.5 weeks of HFD feeding with FTY720-treatment or not. (C) Correlations of mRNA expression levels of CD4⁺ T cells with TGF-β and GITR. Amounts of cDNA were all normalized using TATA box binding protein and hypoxanthine phosphoribosyl transferase. The relative quantity of mRNA was compared to the No HFD group. Data are mean ± SEM. * p<0.05 vs No HFD; ** p<0.05 vs Control. GITR = glucocorticoid induced tumor necrosis factor receptor related gene. HFD = high fat high cholesterol diet.