Apoptotic Ablation of Platelets Reduces Atherosclerosis in Mice With Diabetes

Abstract

Objective: People with diabetes are at a significantly higher risk of cardiovascular disease, in part, due to accelerated atherosclerosis. Diabetic subjects have increased number of platelets that are activated, more reactive, and respond suboptimally to antiplatelet therapies. We hypothesized that reducing platelet numbers by inducing their premature apoptotic death would decrease atherosclerosis. Approach and Results: This was achieved by targeting the antiapoptotic protein Bcl-x_L (B-cell lymphoma-extra large; which is essential for platelet viability) via distinct genetic and pharmacological approaches. In the former, we transplanted bone marrow from mice carrying the Tyr15 to Cys loss of function allele of Bcl-x (known as Bcl-x^Plt20) or wild-type littermate controls into atherosclerotic-prone Ldlr^+/- mice made diabetic with streptozotocin and fed a Western diet. Reduced Bcl-x_L function in hematopoietic cells significantly decreased platelet numbers, exclusive of other hematologic changes. This led to a significant reduction in atherosclerotic lesion formation in Bcl-x^Plt20 bone marrow transplanted Ldlr^+/- mice. To assess the potential therapeutic relevance of reducing platelets in atherosclerosis, we next targeted Bcl-x_L with a pharmacological strategy. This was achieved by low-dose administration of the BH3 (B-cell lymphoma-2 homology domain 3) mimetic, ABT-737 triweekly, in diabetic Apoe^-/- mice for the final 6 weeks of a 12-week study. ABT-737 normalized platelet numbers along with platelet and leukocyte activation to that of nondiabetic controls, significantly reducing atherosclerosis while promoting a more stable plaque phenotype.

Conclusions: These studies suggest that selectively reducing circulating platelets, by targeting Bcl-x_L to promote platelet apoptosis, can reduce atherosclerosis and lower cardiovascular disease risk in diabetes. Graphic Abstract: A graphic abstract is available for this article.

Keywords: atherosclerosis; bone marrow; cardiovascular disease; leukocyte; mice.

Figure 1:. Genetic loss of function in Bcl-x_L decreases platelets in diabetic Ldlr^+/− mice.

A) Experimental overview: Ldlr^+/− mice were transplanted with BM from Bcl-x^PLT20 mice or littermate controls (WT), made diabetic with STZ and fed a WTD. B) Blood glucose and C) plasma cholesterol levels at the end of the study. D) Circulating platelets, E) leukocytes and F) BM haematopoietic stem and progenitor cells were quantified by flow cytometry. Data are presented mean ± SEM and analyzed using a Student’s unpaired t-test, biological replicates of n=6 & 7/group; *p<0.05, ***p<0.001, ****p<0.0001.

Figure 2:. Genetically induced platelet apoptosis decreases atherosclerosis in diabetic Ldlr^+/− mice.

A) Atherosclerosis was quantified in the aortic arch by Oil-Red-O staining and en face analysis. In the aortic sinus, plaques were assessed by B) H&E (size; scale bars = 100 μm), C) Oil-Red-O (lipid), D) CD68⁺ area (macrophages) and E) picrosirius red (collagen), scale bars: 50 μM. Data are presented mean ± SEM and analyzed using a Student’s unpaired t-test, biological replicates of n=6 & 7/group; *p<0.05, **p<0.01, ****p<0.0001.

Figure 3:. Genetically induced platelet apoptosis results in fewer platelet-leukocyte interactions and reduced leukocyte activation.

A) Platelet-leukocyte interactions were assessed by flow cytometry. Ly6-C^hi monocyte (M) and neutrophil (N) populations, identified as per the methods, were overlayed on the same FACS plot for visualization purpose. B) Leukocyte activation (cell-surface CD11b expression) on the platelet-interacting cells was quantified by flow cytometry. Data are presented mean ± SEM and analyzed using a Student’s unpaired t-test, biological replicates of n=6 & 7/group; *p<0.05, **p<0.01, ****p<0.0001.

Figure 4:. Low dose ABT-737 normalizes platelets in diabetic Apoe^−/− mice.

A) Experimental overview: Apoe^−/− mice were made diabetic (STZ) or left as control and fed a standard laboratory diet for 12 weeks. After 6 weeks, a group of the diabetic mice were treated with ABT-737 (30mg/kg/tri-weekly i.p.) for additional 6 weeks. B) Blood glucose (n=7/group) and C) plasma cholesterol levels were measured at the end of the study (n=7, 7, 5/group). Circulating D) platelets, E) percentage and F) number of reticulated platelets (n=7/group), and G) leukocytes were measured by flow cytometry (n=5-6/group). H) BM Haematopoietic stem and progenitor cells were quantified by flow cytometry (n=6-7/group). Data are presented mean ± SEM and analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparisons test, biological replicates n=5-7/group; *p<0.05, **p<0.01, ****p<0.0001 c.f. control, or as indicated.

Figure 5:. ABT-737-induce platelet apoptosis in diabetic Apoe^−/− mice and reduce atherosclerosis.

Atherosclerotic plaques were assessed in the aortic sinus. A) Representative images of lesion size and lipid composition with ORO staining; B) lesion size and C) lipid positive area. D) CD68⁺ area (macrophages) and E) picrosirius red (collagen), scale bars: 50 μM. F) Platelet-leukocyte interactions and G) Ly6-C^hi monocyte activation, as assessed by CD11b levels on the platelet-interacting cells, were quantified by flow cytometry. H) CD62P (P-selectin) expression was assessed on reticulated and mature platelets by flow cytometry. Data are presented mean ± SEM and analyzed using a one-way ANOVA followed by a Dunnett’s multiple comparisons test, biological replicates, n=7, 7, 6/group; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 c.f. control, or as indicated.