IL-1ß Inhibition Partially Negates the Beneficial Effects of Diet-Induced Atherosclerosis Regression in Mice.

BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Secreted Protein Profiling of Human Aortic Smooth Muscle Cells Identifies Vascular Disease Associations.

BACKGROUND: Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. METHODS: Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. RESULTS: We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. CONCLUSIONS: Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

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

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.

Secreted protein profiling of human aortic smooth muscle cells identifies vascular disease associations.

Background: Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular diseases (CVD), the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a "contractile" to a "synthetic" phenotype characterized by an increased proliferation, migration, production of extracellular matrix (ECM) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of CVD, including coronary artery disease (CAD), stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies (GWAS). Methods: Using human aortic SMCs from 123 multi-ancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis of the conditioned media. Results: We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping (pQTL) and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. Conclusions: Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.

IL-1ß inhibition partially negates the beneficial effects of diet-induced lipid lowering.

Background: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions are the leading cause of death in the world. The most common and effective means to reduce these major adverse cardiovascular events (MACE), including myocardial infarction (MI) and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, little is known regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. Methods: Smooth muscle cell (SMC)-lineage tracing Apoe-/- mice were fed a Western diet (WD) for 18 weeks and then switched to a low-fat chow diet for 12 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery (BCA) lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of SMC, and other lesion cells by SMC-lineage tracing combined with scRNA-seq, CyTOF, and immunostaining plus high resolution confocal microscopic z-stack analysis. In addition, to determine if treatment with a potent inhibitor of inflammation could augment the benefits of chow diet-induced reductions in LDL-cholesterol, SMC-lineage tracing Apoe-/- mice were fed a WD for 18 weeks and then chow diet for 12 weeks prior to treating them with an IL-1ß or control antibody (Ab) for 8-weeks. Results: Lipid-lowering by switching Apoe-/- mice from a WD to a chow diet reduced LDL-cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß Ab treatment resulted in multiple detrimental changes including increased plaque burden, BCA lesion size, as well as increased cholesterol crystal accumulation, intra-plaque hemorrhage, necrotic core area, and senescence as compared to IgG control Ab treated mice. Furthermore, IL-1ß Ab treatment upregulated neutrophil degranulation pathways but down-regulated SMC extracellular matrix pathways likely important for the protective fibrous cap. Conclusions: Taken together, IL-1ß appears to be required for chow diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.

Treatment of advanced atherosclerotic mice with the senolytic agent ABT-263 is associated with reduced indices of plaque stability and increased mortality.

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 they are the major source of senescent cells. Moreover, there are no studies of the effect of ABT-263 on endothelial cells (EC), which along with SMC comprise 90% of α-SMA+ myofibroblast-like cells in the protective fibrous cap. Here we tested the hypothesis that treatment of advanced atherosclerotic mice with the 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 WD for 18 weeks, followed by ABT-263 100mg/kg/bw for six weeks or 50mg/kg/bw for nine 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 increased mortality by >50%. Contrary to expectations, treatment of WD-fed Apoe-/- mice with the senolytic agent ABT-263 resulted in multiple detrimental changes including reduced indices of stability, and increased mortality.