Abstract:Atherogenic remodeling often occurs at arterial locations with disturbed blood flow (i.e., low or oscillatory) and both aging and western diet (WD) increase the likelihood for pro-atherogenic remodeling. However, it is unknown if old age and/or a WD modify the pro-atherogenic response to disturbed blood flow. We induced disturbed blood flow by partial carotid ligation (PCL) of the left carotid artery in young and old, normal chow (NC) or WD fed male B6D2F1 mice. Three weeks post-PCL, ligated carotid arteries h… Show more
“…These observations are consistent with numerous reports demonstrating anti-atherosclerotic effects of pharmacological mTORC1-inhibition in mice. However, it has to be considered that, unlike our murine disease model eliciting only atherogenic remodeling and initial atherosclerotic lesions, these studies investigated more advanced stages of atherosclerosis in atheroprone mouse strains lacking the LDL receptor or the apolipoprotein E. While these genetic mouse models may better mimic the complete picture of human atherosclerosis, the lifelong defect in lipid homeostasis resulting in excessive hypercholesterolemia might also be a confounding factor 29 . Indeed, findings from previous studies suggest that mTORC1-inhibition is less effective in late stages of atherosclerosis and that potential effects are obscured by the excessive hypercholesterolemia in genetic atherosclerosis models 7 .…”
Section: Discussionmentioning
confidence: 99%
“…6a) 28 . In combination with sustained western diet, this disturbed blood flow results in pro-inflammatory endothelial activation and atherogenic vascular remodeling in terms of neointimal hyperplasia and immune cell infiltration within four weeks 29 . Even though not observed consistently, initial atherosclerotic lesions with isolated foam cells and intracellular lipid accumulation can also develop.Figure 6Endothelial-specific PRAS40 deficiency promotes atherogenic remodeling in vivo .…”
Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.
“…These observations are consistent with numerous reports demonstrating anti-atherosclerotic effects of pharmacological mTORC1-inhibition in mice. However, it has to be considered that, unlike our murine disease model eliciting only atherogenic remodeling and initial atherosclerotic lesions, these studies investigated more advanced stages of atherosclerosis in atheroprone mouse strains lacking the LDL receptor or the apolipoprotein E. While these genetic mouse models may better mimic the complete picture of human atherosclerosis, the lifelong defect in lipid homeostasis resulting in excessive hypercholesterolemia might also be a confounding factor 29 . Indeed, findings from previous studies suggest that mTORC1-inhibition is less effective in late stages of atherosclerosis and that potential effects are obscured by the excessive hypercholesterolemia in genetic atherosclerosis models 7 .…”
Section: Discussionmentioning
confidence: 99%
“…6a) 28 . In combination with sustained western diet, this disturbed blood flow results in pro-inflammatory endothelial activation and atherogenic vascular remodeling in terms of neointimal hyperplasia and immune cell infiltration within four weeks 29 . Even though not observed consistently, initial atherosclerotic lesions with isolated foam cells and intracellular lipid accumulation can also develop.Figure 6Endothelial-specific PRAS40 deficiency promotes atherogenic remodeling in vivo .…”
Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.
“…Oscillatory shear stress-mediated atherosclerotic lesions were induced in the left carotid artery by partial carotid ligation (PCL). PCL is a surgical procedure that induces advanced human-like atherosclerotic lesions in atheroprone mouse models [ 21 , 22 ]. Briefly, under isoflurane (1–2%) anesthesia, the left carotid artery was exposed by blunt dissection.…”
Section: Methodsmentioning
confidence: 99%
“…Confluent cells were used functional experiments. Primary vascular smooth muscle cells were isolated from aorta as previously described [ 22 ]. Briefly, the thoracic aorta was cleaned of perivascular fatty tissue and digested in an enzyme solution (HBSS buffer with 1 mg/ml collagenase II, 1 mg/ml soybean trypsin Inhibitor, 0.744 unit/ml elastase, and 1% penicillin/streptomycin) for 10 minutes.…”
Section: Methodsmentioning
confidence: 99%
“…However, mouse atherosclerotic plaques are spontaneous and simple in nature, while humans develop advanced and complex lesions. To overcome this limitation, we performed partial carotid ligation (PCL) of the left carotid artery [ 19 , 21 , 22 ]. PCL is a surgical model of induced, acute, oscillatory sheer that, when combined with consumption of an atherogenic diet in ApoE -/- mice, leads to the development of advanced atherosclerotic lesions within 2–5 weeks [ 21 ].…”
Atherosclerosis is the root cause of major cardiovascular diseases (CVD) such as myocardial infarction and stroke. ADP-ribosylation factor 6 (Arf6) is a ubiquitously expressed GTPase known to be involved in inflammation, vascular permeability and is sensitive to changes in shear stress. Here, using atheroprone, ApoE-/- mice, with a single allele deletion of Arf6 (HET) or wildtype Arf6 (WT), we demonstrate that reduction in Arf6 attenuates atherosclerotic plaque burden and severity. We found that plaque burden in the descending aorta was lower in HET compared to WT mice (p˂0.001) after the consumption of an atherogenic Paigen diet for 5 weeks. Likewise, luminal occlusion, necrotic core size, plaque grade, elastic lamina breaks, and matrix deposition were lower in the aortic root atheromas of HET compared to WT mice (all p≤0.05). We also induced advanced human-like complex atherosclerotic plaque in the left carotid artery using partial carotid ligation surgery and found that atheroma area, plaque grade, intimal necrosis, intraplaque hemorrhage, thrombosis, and calcification were lower in HET compared to WT mice (all p≤0.04). Our findings suggest that the atheroprotection afforded by Arf6 heterozygosity may result from reduced immune cell migration (all p≤0.005) as well as endothelial and vascular smooth muscle cell proliferation (both p≤0.001) but independent of changes in circulating lipids (all p≥0.40). These findings demonstrate a critical role for Arf6 in the development and severity of atherosclerosis and suggest that Arf6 inhibition can be explored as a novel therapeutic strategy for the treatment of atherosclerotic CVD.
Using multiple mouse models, we explored the impact of aging on the size and severity of atherosclerotic lesions. In young, middle-aged and old apolipoprotein E knockout mice (ApoE−/−) fed an atherogenic diet (AD) for 3–8 weeks, plaque/atheroma formation in the descending aorta and aortic root, and atheroma development in the carotid in response to partial carotid ligation (PCL) were assessed. Total and LDL cholesterol, and triglycerides were higher in old compared to both other age groups, regardless of AD duration. Aortic plaque burden increased with AD duration in all ages. The size and plaque morphology grade of aortic root atheromas was higher with age; however, there was no effect of age on the size or severity of carotid atheromas after PCL. We additionally induced hyperlipidemia in young and old C57BL/6 mice by adeno-associated virus mediated upregulation of LDL receptor regulator, Pcsk9, and 5 weeks of AD. Despite lower cholesterol in old compared to young Pcsk9 mice, there was a greater size and severity of aortic root atheromas in old mice. However, like the ApoE−/− mice, there was no effect of age on size or severity of PCL-induced carotid artery atheromas in Pcsk9 mice. Together, these results suggest that aging increases the size and severity of spontaneous aortic atheromas.
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