Treatment of advanced atherosclerotic mice with ABT-263 reduced indices of plaque stability and increased mortality
Santosh Karnewar, Vaishnavi Karnewar, Laura S. Shankman, Gary K. Owens
Santosh Karnewar, Vaishnavi Karnewar, Laura S. Shankman, Gary K. Owens
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Research Article Vascular biology

Treatment of advanced atherosclerotic mice with ABT-263 reduced indices of plaque stability and increased mortality

Abstract

The use of senolytic agents to remove senescent cells from atherosclerotic lesions is controversial. A common limitation of previous studies is the failure to rigorously define the effects of senolytic agent ABT-263 (Navitoclax) on smooth muscle cells (SMC) despite studies claiming that these cells are the major source of senescent cells. Moreover, there are no studies on the effect of ABT-263 on endothelial cells (EC), which — along with SMC — comprise 90% of α-smooth muscle actin+ (α-SMA+) myofibroblast-like cells in the protective fibrous cap. Here we tested the hypothesis that treatment of advanced atherosclerotic mice with ABT-263 will reduce lesion size and increase plaque stability. SMC (Myh11-CreERT2-eYFP) and EC (Cdh5-CreERT2-eYFP) lineage tracing Apoe–/– mice were fed a western diet (WD) for 18 weeks, followed by ABT-263 at 100 mg/kg/bw for 6 weeks or 50 mg/kg/bw for 9 weeks. ABT-263 treatment did not change lesion size or lumen area of the brachiocephalic artery (BCA). However, ABT-263 treatment reduced SMC by 90% and increased EC contributions to lesions via EC-to-mesenchymal transition (EndoMT) by 60%. ABT-263 treatment also reduced α-SMA+ fibrous cap thickness by 60% and was associated with a > 50% mortality rate. Taken together, ABT-263 treatment of WD-fed Apoe–/– mice with advanced lesions resulted in multiple detrimental changes, including reduced indices of stability and increased mortality.

Authors

Santosh Karnewar, Vaishnavi Karnewar, Laura S. Shankman, Gary K. Owens

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Figure 5

ABT-263 treatment (100mg/kg/bw) of SMC (Myh11-CreERT2-eYFP) and EC (Cdh5-CreERT2-eYFP) lineage tracing Apoe–/– mice with advanced atherosclerotic lesions was associated with a marked increase in nonsenescent endothelial cell apoptosis.

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ABT-263 treatment (100mg/kg/bw) of SMC (Myh11-CreERT2-eYFP) and EC (Cdh5...
(A) Representative confocal images of costaining for eYFP (for detecting SMC), p21 (detecting senescent cells), TUNEL (detecting apoptotic cells), and DAPI in advanced BCA lesions from SMC lineage tracing Apoe–/–mice were fed a WD for 18 weeks, followed by 100 mg/kg/bw ABT-263 treatment on WD for 6 weeks. The confocal images show a maximum intensity projection ×20 zoom. Scale bar: 100 μm and 20 μm (zoomed-in images). (B) Representative confocal images of costaining for eYFP (for detecting EC), α-SMA+, and DAPI in advanced BCA lesions from EC lineage tracing Apoe–/– mice were fed a WD for 18 weeks followed by 100 mg/kg/bw ABT-263 treatment on WD for 6 weeks. Scale bars: 100 μm (top); 20 μm (bottom). (C) Quantification of the percentage of p21+ (p21+/DAPI+) cells in the fibrous cap from A and B. (D) Quantification of the percentage of TUNEL+ (TUNEL+/DAPI+) cells in the fibrous cap from A and B. (E) Non–SMC-derived nonsenescent apoptotic cells (Myh11-eYFP– p21– TUNEL+/TUNEL+) of all apoptotic cells from A. (F) EC-derived nonsenescent apoptotic cells (Cdh5-eYFP+ p21– TUNEL+/TUNEL+) of all apoptotic cells from B. Mann-Whitney U tests were used for statistical analysis in C–F. Biologically independent animals are indicated as individual dots. Data are shown as mean ± SEM (C and D) and ± SD (E and F). The P values are indicated on the respective graphs.